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Virological Institute of the University Hospital Erlangen, Clinical and Molecular Virology, University of Erlangen-Nuremberg, 91054 Erlangen, Germany
Correspondence
Manfred Marschall
manfred.marschall{at}viro.med.uni-erlangen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table showing oligonucleotides used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
HCMV replication is restricted to specific host-cell types and is dependent on the balance of interactions between viral and cellular proteins. One of the main regulatory processes during virus infection is the intracellular trafficking of viral proteins and particles. The exchange between nucleus and cytoplasm is mediated mainly through the nuclear pore complex, and thus the integrity of the nuclear envelope, which is composed of both membrane and lamina constituents, is crucial for intracellular transport pathways. The nuclear lamina, which lies beneath the inner nuclear membrane, contains a variable number of lamin isoforms and forms a rigid, proteinaceous meshwork. During infection with herpesviruses, the nuclear lamina restricts the efficiency of nucleocytoplasmic transport of viral capsids, as the large size of herpesviral capsids (
120 nm) does not allow for transit through nuclear pores (39 nm; Pante & Kann, 2002). Lamina destabilization requires site-specific phosphorylation of lamins and lamin-binding membrane proteins. Phosphorylation leads to lamin depolymerization and may also permit their release from lamin-binding membrane proteins, including the lamin B receptor (LBR) (Peter et al. 1990; Goldman et al., 2002). Protein kinase C (PKC) and cdc2 have been identified as kinases capable of phosphorylating lamins during mitosis (Peter et al. 1990; Collas et al., 1997). In HCMV-infected cells, in addition to cellular protein kinases, the viral kinase pUL97 also possesses lamin-phosphorylating activity (Marschall et al., 2005). pUL97 has a number of functions within the viral replication cycle and is a target for antiviral drugs (Prichard et al., 1999; Biron et al., 2002; Marschall et al., 2002; Wang et al., 2003; Herget et al., 2004; Swan et al., 2007). pUL97 has been implicated in the nuclear egress of HCMV (Wolf et al., 2001; Krosky et al., 2003; Marschall et al., 2005).
Herpesviruses encode a conserved group of lamina-associated proteins, some of which recruit cellular as well as viral protein kinases to the nuclear lamina (Muranyi et al., 2002; Kato et al., 2006) and seem to be components of a functional nuclear egress complex (Sanchez & Spector, 2002). In particular, the herpes simplex virus 1 (HSV-1)-encoded proteins UL34 and UL31 have been described as essential factors for primary envelopment and thus for nuclear capsid export (Reynolds et al., 2004). A similar functional role has been proposed for their mouse cytomegalovirus (MCMV) (pM50 and pM53) and Epstein–Barr virus (EBV) (BFRF1 and BFLF2) counterparts (Muranyi et al., 2002; Bubeck et al., 2004; Lake & Hutt-Fletcher, 2004; Farina et al., 2005; Gonnella et al., 2005; Lötzerich et al., 2006). However, relatively little information is available for the HCMV counterparts, pUL50 and pUL53. Dal Monte et al. (2002) described a lamina association of pUL53 in infected human fibroblasts. pUL53 co-localized with lamin B and was incorporated into virion tegument. These results are consistent with pUL53 having a role in nucleocapsid maturation, or egress of nucleocapsids from the nucleus to the cytoplasm (Dal Monte et al., 2002).
In this study, we analysed pUL50 and pUL53 expression in DNA transfection experiments to investigate their intracellular localization, protein interactions and whether pUL50/pUL53 complexes mediated recruitment of cellular proteins in a manner similar to that described for their homologues in other herpesviruses.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition to full-length UL50, a truncated version, encoding aa 1–358, was generated. PCR amplification with primers carrying tag sequences resulted in fusion of the ORFs to C-terminal haemagglutinin (HA) or FLAG tags. PCR was performed using Vent DNA polymerase (New England BioLabs) for 35 cycles (denaturation for 40 s at 95 °C, annealing for 40 s at 50 °C and polymerization for 120 s at 72 °C). PCR products were inserted into the vectors pcDNA3.1 (Invitrogen), pGBT9 and pGAD424 (both Clontech) after cleavage with the restriction enzymes EcoRI/XhoI, XhoI/PstI or EcoRI/SalI, respectively. Constructs pDsRed1-N1 and peGFP-N1 expressing red (RFP) or (GFP) green fluorescent protein, respectively, were purchased from BD Clontech and used as positive controls for transfection experiments.
Oligonucleotides.
Oligonucleotide primers used for PCR were purchased from Biomers; their sequences are given in Supplementary Table S1, available in JGV Online.
Cell culture and transfection.
293T and HeLa cells were cultivated in Dulbecco's minimal essential medium containing 10 % fetal calf serum. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen) according to the protocol of the manufacturer, using 70–90 % confluent cells, with a seeding cell number of 4.2x105 (293T) or 3.5x105 (HeLa) cells for six-well plates or 5x106–6x106 (293T) cells for 10 cm dishes.
Indirect immunofluorescent double staining.
HeLa cells were grown on cover slips for transfection. At 2 days post-transfection, cells were fixed with 4 % paraformaldehyde (10 min, room temperature) and permeabilized using PBS/0.2 % Triton X-100 (20 min, 4 °C). Primary antibodies were incubated for 90 min at 37 °C. The secondary antibodies used for double staining were FITC-conjugated (green fluorescence; Dianova) and Cy3-conjugated (red fluorescence; Dianova), and were incubated for 45 min at 37 °C. The nucleus was counterstained with DAPI Vectashield mounting medium (Vector Laboratories). Data for immunofluorescence were collected using an Axiovert-135 microscope (Zeiss) at magnifications of x400 and x630.
Co-immunoprecipitation assay (CoIP).
293T cells were transfected in six-well plates or 10 cm dishes. At 2 days post-transfection, cells were lysed in 500–1000 µl CoIP buffer [50 mM Tris/HCl (pH 8.0), 150–300 mM NaCl, 5 mM EDTA, 0.5 % NP-40, 1 mM PMSF, 2 µg aprotinin ml–1, 2 µg leupeptin ml–1 and 2 µg pepstatin ml–1) and used for CoIP with 1 µl (six-well plate) or 2.5 µl (10 cm dish) of anti-HA or pre-immune rabbit antiserum (anti-HA.11; HISS Diagnostics) for 2 h at 4 °C under rotation. Protein A–Sepharose beads were added to the CoIP reactions (2.5 mg, 2 h at 4 °C; Amersham Pharmacia Biotech). The precipitates were pelleted and washed before the samples were subjected to a standard Western blot analysis using mAbs specific for FLAG (M2; Sigma), PKC
(A-3; Santa Cruz) or GFP (clones 7.1/13.1; Roche) for the detection of co-immunoprecipitates (ECL staining; New England Bio-Laboratories).
In vitro kinase assay (IVKA).
The kinase activity of PKC–GFP was determined in vitro (2.5 µCi of [
-33P]ATP) after immunoprecipitation of the kinase from transfected 293T cells as described previously (Marschall et al., 2001). Putative substrate proteins, such as pUL50(1–358)–HA, were co-expressed with PKC–GFP and co-immunoprecipitated. CoIP was performed in CoIP buffer as described above. The co-immunoprecipitates were pelleted and washed (using IVKA buffer without phosphatidylserine and diacylglycerol) before the samples were subjected to an IVKA [IVKA buffer: 20 mM HEPES (pH 7.4), 0.03 % Triton X-100, 0.1 mg phosphatidylserine ml–1, 10 µg diacylglycerol ml–1 and 10 mM magnesium acetate). Purified histone 2B (H2B; Roche) was added exogenously to the reaction at a concentration of 15 µM. In control settings, staurosporine (STP) was added at a concentration of 1 µM to the IVKA as an inhibitor of PKC activity.
Yeast two-hybrid screening.
Protein interactions were analysed using GAL4 fusion proteins in a yeast two-hybrid system (Fields & Song, 1989). Saccharomyces cerevisiae strain Y153 expressing the proteins pUL50, pUL53 or others (in fusion with GAL4-BD as bait) was used for interaction analysis with selected expression clones (putative interactors in fusion with GAL4-AD, or vice versa) (Durfee et al., 1993). Selection for the presence of bait and interactor plasmids was achieved by cultivation on medium restricting growth to combined tryptophan/leucine prototrophy. Transformants were analysed for 
-galactosidase (-gal) activity by filter lift tests.
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RESULTS |
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47 or 45 kDa, respectively, plus a number of additional faster-migrating species (Fig. 1a). pUL97 was included as a positive control for expression. Four specific protein modifications were reproducibly detectable for pUL53 (Fig. 1a, lanes 2 and 6, indicated by brackets), although the specific mode of modification is unknown. However, we have observed phosphorylation of both pUL53 and pUL50 by inorganic 33P in vivo-labelling of transfected cells (data not shown). Analysis of the primary sequence of the two proteins performed in silico (TMHMM server version 2.0; http://www.cbs.dtu.dk/services/TMHMM/) predicted a putative transmembrane domain in pUL50 at aa 359–381 (Fig. 1b). For pUL53, no region with a probability of membrane localization was detected. To investigate intracellular localization, single transfections and co-transfections of the expression constructs were performed in HeLa cells. Both proteins were detectable exclusively in the nucleus. A putative nuclear localization signal (NLS) of ORF UL53 was predicted for aa 13–24 (PredictNLS server; http://cubic.bioc.columbia.edu/predictNLS/), but this prediction awaits experimental confirmation. No NLS was predictable for ORF UL50. Interestingly, the expressed pUL50 showed a very pronounced association with the nuclear envelope (nuclear rim staining; Fig. 1c, panels a–d), whereas pUL53 appeared to be distributed evenly throughout the nucleus (Fig. 1c, panels e–h). Importantly, upon co-expression of the two proteins, pUL53 relocalized almost completely towards the distinct nuclear rim staining of pUL50 (Fig. 1c, panels i–m). Thus, pUL50 was clearly dominant, altering the nuclear localization of pUL53. A deletion mutant of pUL50 lacking the predicted transmembrane domain [pUL50(1–358), Fig. 1c, panel o] exhibited a diffuse nuclear distribution and failed to influence the localization of pUL53 (Fig. 1c, panels n–q). Thus, the capacity of pUL50 to induce relocalization of pUL53 is dependent on the association of pUL50 with the nuclear envelope. Co-staining of pUL50 with endogenous lamins A/C showed a defined co-localization (Fig. 1c, panels r–u), consistent with pUL50 being associated with the inner nuclear membrane and/or the nuclear lamina.
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strongly implied that there is a direct interaction between pUL50 and pUL53. This potential interaction was therefore investigated in a yeast two-hybrid assay. Full-length pUL50 (including the transmembrane domain) was analysed initially, but appeared to be non-functional in the yeast two-hybrid system, probably due to its membrane-binding properties (data not shown). When a C-terminally truncated version of pUL50 was used [pUL50(1–358)], an interaction between pUL50(1–358) and pUL53 was detected (in both directions; Fig. 2a), albeit at a low level of signal intensity compared with a standard positive control (Fig. 2b, top panel). In control settings, neither protein produced any background activity (vector controls; Fig. 2b) and, moreover, did not interact with themselves (Fig. 2a, top two panels). Additional reactivity was observed between pUL50(1–358) and cellular proteins known to be transiently associated with the nuclear lamina, p32 and PKC (isoforms 
and 
). p32 is known to interact with a range of proteins including LBR (Mylonis et al., 2004), PKC (Storz et al., 2000; Robles-Flores et al., 2002) and cytomegaloviral pUL97 (Marschall et al., 2005). Addressing these issues of specificity, p32 did not interact with a series of other cytomegaloviral proteins including pUL53 (data not shown). pUL50, on the other hand, showed specific interactions with pUL53, p32 and PKC, but not with an N-terminal fragment of LBR (aa 1–208) or with pUL97 (Fig. 2a). Furthermore, several known interaction pairs could be reproduced in this series of experiments, i.e. the interactions of p32 with PKC, pUL97 with p32 and LBR with p32 (Fig. 2a). No further interactions were detected for pUL53. Thus, a higher complex composed of viral and cellular lamina-associated proteins may be postulated according to the multifold interactions identified with the yeast two-hybrid system. This analysis indicates that pUL50 is a multifold interactor protein binding directly to pUL53, p32 and PKC.
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). In CoIP experiments, a strong positive signal was obtained for the interaction between pUL50(1–358) and pUL53 (Fig. 3a), whilst interaction of pUL50(1–358) with another cytomegaloviral protein, pUL97, was negative. Using the full-length version of pUL50 in a parallel CoIP setting, an interaction with co-expressed pUL53 was also detectable, but the signal intensity was lower (possibly due to the loss of an insoluble membrane-bound fraction of full-length pUL50; data not shown). In addition, the interaction between pUL50 and cellular PKC was confirmed (Fig. 3c). Using a GFP-fused construct of PKC, CoIP was positive for pUL50, but negative for pUL53 and pUL97 tested in parallel (Fig. 3c, lanes 3–5). When Western blot detection of the CoIP samples was performed with a mAb recognizing both recombinant and endogenous PKC, two PKC-specific bands were obtained, with the recombinant form being dominant over a faint band for endogenous PKC (Fig. 3c, lane 8).
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–GFP were expressed separately, immunoprecipitated and added to IVKA reactions (twofold excess of substrate precipitates compared with kinase precipitates). Numerous weakly phosphorylated protein species were detected that potentially co-migrated with pUL50–HA and pUL53–HA, but these did not allow a conclusive interpretation (data not shown). Therefore, an improved setting was performed using a combined CoIP/IVKA strategy. PKC and putative substrates, such as pUL50(1–358)–HA, were co-expressed with PKC–GFP and co-immunoprecipitated using a tag-specific antibody (anti-HA; Fig. 4a). In this case, clear phosphorylation of pUL50(1–358)–HA by PKC–GFP was detectable (Fig. 4a, lanes 5 and 7). pUL53–HA did not co-immunoprecipitate PKC–GFP (Fig. 4b, upper panel, lanes 6 and 9); thus, no signals for phosphorylation of pUL53–HA by PKC–GFP were obtained (Fig. 4a, lane 6). The co-immunoprecipitation of PKC–GFP by pUL50(1–358)–HA was clearly detectable (Fig. 4b, lower panel, lanes 5, 7 and 8). Autophosphorylation of PKC–GFP (which was not chemically stimulated in transfected cells) was below the threshold of detection (Fig. 4a, lanes 5 and 7). The strongest signals for in vitro phosphorylation of the standard substrate H2B were demonstrated for those samples containing PKC–GFP (Fig. 4a, lanes 5 and 7). Other detectable signals for H2B phosphorylation most probably resulted from the co-immunoprecipitation of endogenous PKC or an unknown associated protein kinase. As a specificity control, a known inhibitor of PKC activity, STP (1 µM), was applied to the reactions, resulting in complete suppression of phosphorylation signals for pUL50(1–358)–HA and H2B (Fig. 4a, lanes 8 and 9). Thus, phosphorylation of pUL50 was detectable in vitro and PKC is the most probable candidate for this activity.
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–GFP (Fig. 5, panels a–d) changed to favour a nuclear or perinuclear rim distribution when pUL50 and pUL53 were co-expressed (Fig. 5, panels e–h). Thus, PKC was partially recruited to the nuclear envelope as a consequence of pUL50/pUL53 expression. pUL50 alone, in the absence of co-expressed pUL53, was sufficient to relocalize PKC–GFP (data not shown). Following relocalization, PKC–GFP was observed not only to co-localize with endogenous LBR, but also to be associated with a reduced LBR immunofluorescent signal in individual cells (Fig. 5, panels i–m). Comparable immunofluorescence experiments performed in parallel confirmed the reproducibility of the finding. Thus, the reduction of detectable levels of LBR associated with the co-localization and accumulation of PKC might be the result of a reduction in the integrity of the nuclear lamina. From these results, we concluded that the viral protein complex pUL50–pUL53 mediates relocalization of PKC and speculate that this is associated with a disintegrative process in the nuclear lamina. Similar effects have been described for HSV-1 UL34-mediated recruitment of PKC (Park & Baines, 2006).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In prior studies on the HSV-1 homologues of UL50 and UL53, an interaction between UL34 and UL31 has been described (Liang & Baines, 2005). In addition, an interaction between positional homologues of UL34 and UL31 in EBV and MCMV has been demonstrated (Gonnella et al., 2005; Lötzerich et al., 2006). Thus, the interaction between members of the herpesviral UL34/UL31 protein homology group seem to have been conserved during herpesvirus evolution and may be crucial for their function. As it is known that UL34 is a type II transmembrane protein (Purves et al., 1992; Roller et al., 2000; Shiba et al., 2000), we performed a transmembrane prediction for its HCMV homologue, pUL50. A C-terminal region of 23 aa was identified as a potential transmembrane domain. Consistent with this finding, pUL50 localized in a rim pattern around the nuclear envelope in transfected cells. More specifically, an association with the nuclear lamina was illustrated by perfect co-localization of pUL50 with endogenous lamin A/C as well as LBR. In contrast, pUL53 showed diffuse nuclear localization when expressed alone, but was strictly relocalized by pUL50 to a lamina-associated localization. A similar situation has been shown for HSV-1 UL31, which co-localized with UL34 at the nuclear envelope upon co-expression but showed a non-defined nuclear distribution in the absence of other viral proteins (Reynolds et al., 2001).
An important common feature of UL34/UL31 and their homologues is their interaction with viral and cellular protein kinases (Muranyi et al., 2002; Reynolds et al., 2004; Ryckman & Roller, 2004; Bjerke & Roller, 2006; Kato et al., 2006; Park & Baines, 2006). In particular, cellular PKC is strongly recruited by viral lamina-associated proteins. In the present study, a direct interaction between pUL50 and PKC was demonstrated by yeast two-hybrid and CoIP analyses. In agreement with this concept was the finding that co-expressed pUL50 and pUL53 were able to relocalize PKC towards the nuclear lamina. In addition, an in vitro phosphorylation study provided strong evidence that pUL50 is a substrate of PKC.
Taken together, our studies suggest an involvement of pUL50 and pUL53 in cytomegalovirus-induced alterations of the nuclear envelope in the context of nuclear capsid export. We demonstrated properties of pUL50 and pUL53 similar to those of homologues of other herpesviruses, which seem to suggest that pUL50 and pUL53 likewise play a role in the nuclear egress of viral capsids. Importantly, both proteins have been categorized as essential for virus replication in vitro (Dunn et al., 2003; Yu et al., 2003). For MCMV replication, a scenario was based on the formation of a nuclear egress complex composed of cellular and viral proteins including pM50 and pM53 (Muranyi et al., 2002; Bubeck et al., 2004; Rupp et al., 2007), which may be essential for capsid egress. It was postulated that recruitment of specific protein kinases may lead to increased phosphorylation of lamins, resulting in the depolymerization of the nuclear lamina (Muranyi et al., 2002; Sanchez & Spector, 2002; Lötzerich et al., 2006). In the case of HCMV, pUL50 also seems to possess an important recruitment function. In particular, the interaction of pUL50 with PKC and its ability to relocalize PKC to nuclear lamina sites seem to be connected with a PKC-induced reduction in detectable levels of LBR. Moreover, it is known that the cytomegaloviral kinase pUL97 is recruited to the nuclear lamina mainly through its interaction with p32 (Marschall et al., 2005). Thus, our data provide evidence that pUL50 and pUL53 possess highly defined properties of nuclear protein interactions and suggest that the two proteins may have particular importance for the formation of a multicomponent egress complex.
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ACKNOWLEDGEMENTS |
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/, Takuro Arimura (University of Tokyo, Japan) for providing yeast two-hybrid clones pADT7-PKC// and pBKT7-PKC//, H. J. Worman (Columbia University, USA) for providing clone pGBT-LBRAT(1-208) and Paola Dal Monte (University of Bologna, Italy) for providing an anti-UL53 antiserum. This study was supported by Bayerische Forschungsstiftung (grant 576/03), Deutsche Forschungsgemeinschaft (grant MA 1289/4-1) and Johannes und Frieda Marohn-Stiftung, University of Erlangen-Nuremberg (grant FWN-Zo). |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Bjerke, S. L. & Roller, R. J. (2006). Roles for herpes simplex virus type 1 UL34 and US3 proteins in disrupting the nuclear lamina during herpes simplex virus type 1 egress. Virology 347, 261–276.[CrossRef][Medline]
Bubeck, A., Wagner, M., Ruzsics, Z., Lötzerich, M., Iglesias, M., Singh, I. R. & Koszinowski, U. H. (2004). Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication. J Virol 78, 8026–8035.
Collas, P., Thompson, L., Fields, A. P., Poccia, D. L. & Courvalin, J. C. (1997). Protein kinase C-mediated interphase lamin B phosphorylation and solubilization. J Biol Chem 272, 21274–21280.
Curran, M. & Noble, S. (2001). Valganciclovir. Drugs 61, 1145–1150.[CrossRef][Medline]
Dal Monte, P., Pignatelli, S., Zini, N., Maraldi, N. M., Perret, E., Prevost, M. C. & Landini, M. P. (2002). Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein. J Gen Virol 83, 1005–1012.
Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H. & Liu, F. (2003). Functional profiling of a human cytomegalovirus genome. Proc Natl Acad Sci U S A 100, 14223–14228.
Durfee, T., Becherer, K., Chen, P. L., Yeh, S. H., Yang, Y., Kilburn, A. E., Lee, W. H. & Elledge, S. J. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev 7, 555–569.
Farina, A., Feederle, R., Raffa, S., Gonnella, R., Santarelli, R., Frati, L., Angeloni, A., Torrisi, M. R., Faggioni, A. & Delecluse, H. J. (2005). BFRF1 of Epstein–Barr virus is essential for efficient primary viral envelopment and egress. J Virol 79, 3703–3712.
Fields, S. & Song, O. (1989). A novel genetic system to detect protein–protein interactions. Nature 340, 245–246.[CrossRef][Medline]
Fleckenstein, B., Müller, I. & Collins, J. (1982). Cloning of the complete human cytomegalovirus genome in cosmids. Gene 18, 39–46.[CrossRef][Medline]
Goldman, R. D., Gruenbaum, Y., Moir, R. D., Shumaker, D. K. & Spann, T. P. (2002). Nuclear lamins: building blocks of nuclear architecture. Genes Dev 16, 533–547.
Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., Granato, M., Modesti, A., Frati, L. & other authors (2005). Characterization and intracellular localization of the Epstein–Barr virus protein BFLF2: interactions with BFRF1 and with the nuclear lamina. J Virol 79, 3713–3727.
Herget, T., Freitag, M., Morbitzer, M., Stamminger, T. & Marschall, M. (2004). A novel chemical class of pUL97 protein kinase-specific inhibitors with strong anti-cytomegaloviral activity. Antimicrob Agents Chemother 48, 4154–4162.
Kato, A., Yamamoto, M., Ohno, T., Tanaka, M., Sata, T., Nishiyama, Y. & Kawaguchi, Y. (2006). Herpes simplex virus 1-encoded protein kinase UL13 phosphorylates viral Us3 protein kinase and regulates nuclear localization of viral envelopment factors UL34 and UL31. J Virol 80, 1476–1486.
Krosky, P. M., Baek, M. C. & Coen, D. M. (2003). The human cytomegalovirus UL97 protein kinase, an antiviral drug target, is required at the stage of nuclear egress. J Virol 77, 905–914.[CrossRef][Medline]
Lake, C. M. & Hutt-Fletcher, L. M. (2004). The Epstein–Barr virus BFRF1 and BFLF2 proteins interact and coexpression alters their cellular localization. Virology 320, 99–106.[CrossRef][Medline]
Liang, L. & Baines, J. D. (2005). Identification of an essential domain in the herpes simplex virus 1 UL34 protein that is necessary and sufficient to interact with UL31 protein. J Virol 79, 3797–3806.
Lötzerich, M., Ruzsics, Z. & Koszinowski, U. H. (2006). Functional domains of murine cytomegalovirus nuclear egress protein M53/p38. J Virol 80, 73–84.
Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. (2001). Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97. J Gen Virol 82, 1439–1450.
Marschall, M., Stein-Gerlach, M., Freitag, M., Kupfer, R., van den Bogaard, M. & Stamminger, T. (2002). Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle of antiviral therapy. J Gen Virol 83, 1013–1023.
Marschall, M., Marzi, A., aus dem Siepen, P., Jochmann, R., Kalmer, M., Auerochs, S., Lischka, P., Leis, M. & Stamminger, T. (2005). Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina. J Biol Chem 280, 33357–33367.
Muranyi, W., Haas, J., Wagner, M., Krohne, G. & Koszinowski, U. H. (2002). Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina. Science 297, 854–857.
Mylonis, I., Drosou, V., Brancorsini, S., Nikolakaki, E., Sassone-Corsi, P. & Giannakouros, T. (2004). Temporal association of protamine 1 with the inner nuclear membrane protein lamin B receptor during spermiogenesis. J Biol Chem 279, 11626–11631.
Pante, N. & Kann, M. (2002). Nuclear pore complex is able to transport macromolecules with diameters of about 39 nm. Mol Biol Cell 13, 425–434.
Park, R. & Baines, J. D. (2006). Herpes simplex virus type 1 infection induces activation and recruitment of protein kinase C to the nuclear membrane and increased phosphorylation of lamin B. J Virol 80, 494–504.
Peter, M., Nakagawa, J., Doree, M., Labbe, J. C. & Nigg, E. A. (1990). In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell 61, 591–602.[CrossRef][Medline]
Prichard, M. N., Gao, N., Jairath, S., Mulamba, G., Krosky, P., Coen, D. M., Parker, B. O. & Pari, G. S. (1999). A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency. J Virol 73, 5663–5670.
Purves, F. C., Spector, D. & Roizman, B. (1992). UL34, the target of the herpes simplex virus US3 protein kinase, is a membrane protein which in its unphosphorylated state associates with novel phosphoproteins. J Virol 66, 4295–4303.
Reynolds, A. E., Ryckman, B. J., Baines, J. D., Zhou, Y., Liang, L. & Roller, R. J. (2001). UL31 and UL34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids. J Virol 75, 8803–8817.
Reynolds, A. E., Liang, L. & Baines, J. D. (2004). Conformational changes in the nuclear lamina induced by herpes simplex virus type 1 require genes UL31 and UL34. J Virol 78, 5564–5575.
Robles-Flores, M., Rendon-Huerta, E., Gonzalez-Aguilar, H., Mendoza-Hernandez, G., Islas, S., Mendoza, V., Ponce-Castaneda, M. V., Gonzalez-Mariscal, L. & Lopez-Casillas, F. (2002). p32 (gC1qBP) is a general protein kinase C (PKC)-binding protein. J Biol Chem 277, 5247–5255.
Roller, R. J., Zhou, Y., Schnetzer, R., Ferguson, J. & DeSalvo, D. (2000). Herpes simplex virus type 1 UL34 gene product is required for viral envelopment. J Virol 74, 117–129.
Rupp, B., Ruzsics, Z., Buser, C., Adler, B., Walther, P. & Koszinowski, U. H. (2007). Random screening for dominant-negative mutants of the cytomegalovirus nuclear egress protein M50. J Virol 81, 5508–5517.
Ryckman, B. J. & Roller, R. J. (2004). Herpes simplex virus type 1 primary envelopment: UL34 protein modification and the US3–UL34 catalytic relationship. J Virol 78, 399–412.
Sanchez, V. & Spector, D. H. (2002). CMV makes a timely exit. Science 297, 778–779.
Shiba, C., Daikoku, T., Goshima, F., Takakuwa, H., Yamauchi, Y., Koiwai, O. & Nishiyama, Y. (2000). The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment. J Gen Virol 81, 2397–2405.
Storz, P., Hausser, A., Link, G., Dedio, J., Ghebrehiwet, B., Pfizenmaier, K. & Johannes, F. J. (2000). Protein kinase µ is regulated by the multifunctional chaperon protein p32. J Biol Chem 275, 24601–24607.
Swan, S. K., Smith, W. B., Marbury, T. C., Schumacher, M., Dougherty, C., Mico, B. A. & Villano, S. A. (2007). Pharmacokinetics of maribavir, a novel oral anticytomegalovirus agent, in subjects with varying degrees of renal impairment. J Clin Pharmacol 47, 209–217.
Wang, L. H., Peck, R. W., Yin, Y., Allanson, J., Wiggs, R. & Wire, M. B. (2003). Phase I safety and pharmacokinetic trials of 1263W94, a novel oral anti-human cytomegalovirus agent, in healthy and human immunodeficiency virus-infected subjects. Antimicrob Agents Chemother 47, 1334–1342.
Wolf, D. G., Courcelle, C. T., Prichard, M. N. & Mocarski, E. S. (2001). Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation. Proc Natl Acad Sci U S A 98, 1895–1900.
Yu, D., Silva, M. C. & Shenk, T. (2003). Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc Natl Acad Sci U S A 100, 12396–12401.
Received 13 February 2007;
accepted 1 June 2007.
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