| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
Institute of Molecular Biology, Friedrich-Loeffler-Institut, Boddenblick 5A, 17493 Greifswald-Insel Riems, Germany
Correspondence
Axel Karger
axel.karger{at}fli.bund.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). PrV pUL36 function is important for virion morphogenesis in the cytoplasm after nuclear egress. pUL36 interacts with pUL37, another conserved tegument protein (Klupp et al., 2002). However, removal of the N-terminally located pUL37-binding domain does not result in a complete loss of function of pUL36, indicating that the essential requirement for this protein is due to additional, still unknown, functions (Fuchs et al., 2004). Recently, a deubiquitinating proteolytic activity was demonstrated to reside in the very N terminus of herpesvirus pUL36 homologues (Kattenhorn et al., 2005). Thus, both hitherto assigned functional regions of pUL36 reside in the N-terminal part of the protein. It has been shown in a previous study that PrV particles devoid of the tegument proteins pUS3, pUL47 and pUL49 or pUS8 (the envelope glycoprotein E) incorporated the same amounts of several tegument proteins like pUL36, pUL37 and pUL47 as wild-type virions (Michael et al., 2006). This stoichiometric incorporation suggests that, like assembly of the capsid, the architecture of the tegument might, in part, be strictly controlled. On the other hand, amounts of other tegument proteins like pUL46, pUL48 and pUL49 varied to some extent among the different mutants, indicating also some degree of flexibility in tegument composition. The suggested interaction of pUL36 with pentonal sites on the capsid (Zhou et al., 1999), which are devoid of the small capsid protein pUL35 that decorates hexons, may partly explain this strict stoichiometry.
In a study to analyse single-protein-deleted PrV mutants for alterations in the composition of the virion beyond the loss of the deleted protein, structural proteins in virus particles were quantified by a procedure designated ‘stable isotope labelling by amino acids in cell culture’ (SILAC) developed by Ong et al. (2002), which was adapted for virions by our group (Michael et al., 2006).
Virus particles from infected porcine kidney cell cultures were purified by sucrose-density-gradient centrifugation (Karger et al., 1998). Deletion mutants were propagated on cells maintained in DME/F12 cell culture medium (Sigma-Aldrich D-9785) supplemented with conventional leucine, whereas wild-type PrV-Ka (Kaplan & Vatter, 1959) was harvested from cells that had been cultured in medium containing exclusively deuterated leucine (L-leucine-5,5,5-d3; Sigma-Aldrich, 99 atom% D), inserting a mass tag of 3 Da per leucine residue. Mutant and mass-tagged purified PrV-Ka were mixed at 1 : 1 protein ratios and proteins were separated by SDS-PAGE (Laemmli, 1970). Protein bands of interest were cut from Coomassie-stained gels (Neuhoff et al., 1988) and digested with trypsin for peptide mass fingerprint (PMF) analysis (Rosenfeld et al., 1992).
Matrix-assisted laser desorption/ionization time-of-flight mass spectra were registered on an Ultraflex Instrument (Bruker) and data were further processed by FLEXANALYSIS and BIOTOOLS software (Bruker). Database queries were performed with MASCOT (Matrixscience; Perkins et al., 1999) software using an in-house database representing the PrV proteome (Klupp et al., 2004). Quantitative evaluation of the spectra was carried out using in-house software. Mass lists were screened for peak pairs representing leucine-containing pUL36-specific peptides in the conventional and mass-tagged variant and a mean intensity ratio, reflecting the relative amounts of protein originating from wild-type PrV or mutant virions, was calculated from all qualified peaks of one spectrum. Tryptic peptides derived from the major capsid protein, which is present in 960 copies (Steven & Spear, 1997) in every intact virion, were used as an internal standard (Michael et al., 2006). Values above 1.0 indicate an excess of the protein in the mutant particle. Deletion mutants, which were compared with their parental strain PrV-Ka, have been described elsewhere (PrV-
UL11, Kopp et al., 2003; PrV-UL16, PrV-UL21, Klupp et al., 2005a; PrV-UL49, Fuchs et al., 2002; PrV-UL51, Klupp et al., 2005b; PrV-US3, Klupp et al., 2001; PrV-gE, Kopp et al., 2004).
To assay for a potential variation in pUL36 incorporation in different wild-type PrV strains, virions of PrV-Ka, PrV-Becker (Robbins et al., 1984) or PrV-NIA-3 (Baskerville, 1973) were analysed for pUL36 incorporation. The protein migrating at the calculated molecular mass of 330 kDa for the full-length pUL36 (Fig. 1) was identified as pUL36 by PMF and by immunoblot analysis (Towbin et al., 1979) with a pUL36-specific antiserum (Fig. 1b; Klupp et al., 2002). No difference was observed between the three different wild-type PrV strains in the level of incorporation as deduced from the gel or in the size of the packaged pUL36.
|
UL11 and PrV-UL16 virions in Coomassie-stained gels (Fig. 2a). Silver staining with prolonged development (Fig. 2b) revealed that mutants lacking pUS3, pUL21, pUL49, pUL51 or gE contained only minute amounts of proteins migrating in this region of the gel (Fig. 2b). In immunoblot analysis, the additional approximately 220 kDa protein found in PrV-UL11 and PrV-UL16 reacted with a pUL36-specific antiserum (Fig. 2c), but after prolonged film exposure, faint reactivity in this region of the blot was also found in PrV-Ka and the other mutants, indicating that pUL36-derived proteins of the same approximate size were also present, although much less abundantly.
|
UL16 was slightly smaller than that in PrV-UL11, but this size difference did not translate into different peptide coverages in the PMF analysis. Thus, it remains unclear whether the different apparent molecular masses result from additional truncations of pUL36 found in PrV-UL16 virions or from differential post-translational modifications. Although present in PrV-Ka only in very small amounts, relative levels of the pUL36 N-terminal fragments in PrV-UL11 and PrV-UL16 could be determined by the SILAC technique from mixtures of mutant virus particles with mass-tagged PrV-Ka. Levels of the pUL36 N-terminal fragment were elevated 3- to 4-fold in PrV-UL11 and 5- to 6-fold in PrV-UL16. Concomitant with the appearance of the N-terminal pUL36 fragment in the UL11 and UL16 deletion mutants, relative amounts of full-length pUL36 were reduced in both mutants, but were at wild-type levels in all the other mutants tested (Fig. 3). Colorimetric evaluation of the Coomassie-stained gels (AIDA software package; Raytest) confirmed the quantitative MS data. The pUL36 N-terminal fragment was found to represent 26.4 % (PrV-UL11) and 32.9 % (PrV-UL16) of the molar amounts of the respective full-length products and, thus, compensates for a significant part of the mean loss of 37 % (PrV-UL11) and 41 % (PrV-UL16) of the full-length pUL36 (Fig. 3). In all other mutants tested, amounts of the pUL36 N-terminal fragments were minimal and were 1.1–4.0 % of the full-length product.
|
UL11 and PrV-UL16 resulted from overexpression in infected cells, expression levels were assayed by immunoblot analysis of rabbit kidney (RK13) cells infected with PrV-Ka, PrV-UL11 or PrV-UL16 (Fig. 2e
). Cells were extracted 16 h after infection with PBS containing 1 % Triton X-100 and a protease inhibitor cocktail (Complete; Roche) for 10 min on ice with occasional shaking. Twenty micrograms of the clarified extract (15 000 g, 15 min, 4 °C) were analysed after electrophoretic separation on 5 % polyacrylamide gels (Laemmli, 1970). Expression patterns and ratios between the full-length form and the N-terminal fragment (marked by arrows) were similar after infection of RK13 cells with any virus and no excessive expression of the pUL36 N-terminal fragment was observed after infection with PrV-UL11 or PrV-UL16, indicating that incorporation of the pUL36 N-terminal fragment in mutant viruses did not simply reflect its amounts in infected cells. Immunoblot analysis of purified PrV-UL11 or PrV-UL16 virions that had been propagated on pUL11- or pUL16-expressing recombinant cells, respectively (RK13-UL11, RK13-UL16 in Fig. 2d; Kopp et al., 2003; Klupp et al., 2005a) showed that the pUL36 N-terminal fragment was not incorporated into virus particles after phenotypic complementation of PrV-UL11 and PrV-UL16, although it was well expressed in the respective infected cells (Fig. 2e). These results indicate that the presence of pUL11 and pUL16 is required to preclude any pUL36 N-terminal fragment present from incorporation into the mature virion. The origin and potential function of the pUL36 N-terminal fragment is unclear. Since two functional motifs mediating pUL37 interaction and proteolytic deubiquitinating activity are located in the N terminus of pUL36 (Klupp et al., 2002; Fuchs et al., 2004; Kattenhorn et al., 2005), these domains are most likely still functional in the N-terminal pUL36 fragment. Thus, its incorporation into virions could be mediated in the network of protein–protein interactions by its complex formation with pUL37. However, most interesting is the specific correlation of incorporation of the N-terminal pUL36 fragment with the lack of pUL11 and pUL16. In both mutants, incorporation of the N terminus of pUL36 into virions was accompanied by a significant loss of full-length pUL36, suggesting that full-length pUL36 was partly replaced by the N-terminal fragment. This could mean that the requirement for constant amounts of pUL36 in an intact tegument can be accomplished by incorporation of an N-terminal fragment and that the stoichiometric interaction of pUL36 with other virion components is mediated by the N terminus rather than the C terminus. Although neither UL11 nor UL16 is essential for the replication of PrV in cell culture, an impairment in secondary envelopment has been shown in a UL11 mutant (Kopp et al., 2003) in electron microscopy studies. Interestingly, the absence of pUL36 also results in a defect in cytoplasmic virion morphogenesis, whereas deletion of UL36, UL11, UL16 or UL11+UL16 does not influence the intranuclear stages of virus morphogenesis (Fuchs et al., 2004; Klupp et al., 2005a). Loomis et al. (2003) have demonstrated that pUL11 and pUL16 of HSV-1 form a complex and that pUL11 of PrV can interact with pUL16 of HSV-1. Therefore, formation of a complex between pUL11 and pUL16 of PrV seems likely. From the data presented here, it is hypothesized that pUL11 and pUL16 or a potential complex of both proteins play an important role in the exclusive incorporation of full-length pUL36 into the tegument of mature PrV virions excluding any N-terminal fragments of pUL36 that are present in infected cells.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Fuchs, W., Klupp, B. G., Granzow, H., Hengartner, C., Brack, A., Mundt, A., Enquist, L. W. & Mettenleiter, T. C. (2002). Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49. J Virol 76, 8208–8217.
Fuchs, W., Klupp, B. G., Granzow, H. & Mettenleiter, T. C. (2004). Essential function of the pseudorabies virus UL36 gene product is independent of its interaction with the UL37 protein. J Virol 78, 11879–11889.
Kaplan, A. S. & Vatter, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394–407.[CrossRef][Medline]
Karger, A., Bettin, B., Granzow, H. & Mettenleiter, T. C. (1998). Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane. J Virol Methods 70, 219–224.[CrossRef][Medline]
Kattenhorn, L. M., Korbel, G. A., Kessler, B. M., Spooner, E. & Ploegh, H. L. (2005). A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae. Mol Cell 19, 547–557.[CrossRef][Medline]
Klupp, B. G., Granzow, H. & Mettenleiter, T. C. (2001). Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus. J Gen Virol 82, 2363–2371.
Klupp, B. G., Fuchs, W., Granzow, H., Nixdorf, R. & Mettenleiter, T. C. (2002). Pseudorabies virus UL36 tegument protein physically interacts with the UL37 protein. J Virol 76, 3065–3071.
Klupp, B. G., Hengartner, C. J., Mettenleiter, T. C. & Enquist, L. W. (2004). Complete, annotated sequence of the pseudorabies virus genome. J Virol 78, 424–440.
Klupp, B. G., Bottcher, S., Granzow, H., Kopp, M. & Mettenleiter, T. C. (2005a). Complex formation between the UL16 and UL21 tegument proteins of pseudorabies virus. J Virol 79, 1510–1522.
Klupp, B. G., Granzow, H., Klopfleisch, R., Fuchs, W., Kopp, M., Lenk, M. & Mettenleiter, T. C. (2005b). Functional analysis of the pseudorabies virus UL51 protein. J Virol 79, 3831–3840.
Kopp, M., Granzow, H., Fuchs, W., Klupp, B. G., Mundt, E., Karger, A. & Mettenleiter, T. C. (2003). The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm. J Virol 77, 5339–5351.
Kopp, M., Granzow, H., Fuchs, W., Klupp, B. & Mettenleiter, T. C. (2004). Simultaneous deletion of pseudorabies virus tegument protein UL11 and glycoprotein M severely impairs secondary envelopment. J Virol 78, 3024–3034.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Loomis, J. S., Courtney, R. J. & Wills, J. W. (2003). Binding partners for the UL11 tegument protein of herpes simplex virus type 1. J Virol 77, 11417–11424.
Michael, K., Klupp, B. G., Mettenleiter, T. C. & Karger, A. (2006). Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E. J Virol 80, 1332–1339.
Neuhoff, V., Arold, N., Taube, D. & Ehrhardt, W. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.[CrossRef][Medline]
Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A. & Mann, M. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1, 376–386.
Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. (1999). Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567.[CrossRef][Medline]
Robbins, A. K., Weis, J. H., Enquist, L. W. & Watson, R. J. (1984). Construction of E. coli expression plasmid libraries: localization of a pseudorabies virus glycoprotein gene. J Mol Appl Genet 2, 485–496.[Medline]
Rosenfeld, J., Capdevielle, J., Guillemot, J. C. & Ferrara, P. (1992). In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203, 173–179.[CrossRef][Medline]
Steven, A. C. & Spear, P. G. (1997). Herpesvirus capsid assembly and envelopment. In Structural Biology of Viruses, pp. 312–351. Edited by W. Chiu, R. M. Burnett & R. Garcea. New York: Oxford University Press.
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.
Zhou, Z. H., Chen, D. H., Jakana, J., Rixon, F. J. & Chiu, W. (1999). Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions. J Virol 73, 3210–3218.
Received 28 April 2006;
accepted 22 August 2006.
This article has been cited by other articles:
|
|
 |
|
  F. Abaitua and P. O'Hare Identification of a Highly Conserved, Functional Nuclear Localization Signal within the N-Terminal Region of Herpes Simplex Virus Type 1 VP1-2 Tegument Protein J. Virol., June 1, 2008; 82(11): 5234 - 5244. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
