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1 Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, St Orsola General Hospital, Via Massarenti 9, 40138 Bologna, Italy
2 IGM-CNR, Unit of Bologna, c/o IOR, Via di Barbiano 1/10, 40136 Bologna, Italy
Correspondence
Daria Camozzi
daria.camozzi{at}unibo.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Mettenleiter et al., 2006; Severi et al., 1988; Skepper et al., 2001; Stackpole, 1969), which consists of two leaflets, the inner and the outer nuclear membrane (INM and ONM, respectively) separated by the perinuclear space; nuclear capsids acquire a temporary envelope at the INM, which is subsequently lost by fusion with the ONM, allowing access to the cytosol. However, before crossing the nuclear envelope, virions have to overcome a massive obstacle underlying the INM, represented by the nuclear lamina, a thick meshwork of proteins associating in a highly organized structure. The main components of the nuclear lamina are the lamins, which, based on their expression patterns, properties and location, can be divided into two main classes: A-type lamins, including lamin A, A
10, C and C2; and B-type lamins, including lamin B1, B2 and B3 (Broers et al., 1997, 2006; Rzepecki, 2002; Stuurman et al., 1998). Several studies have shown that herpesvirus infection causes structural and biochemical rearrangements of nuclear lamina components, to ensure the efficient production of viral progeny (Leach et al., 2007; Marschall et al., 2005; Morris et al., 2007; Mou et al., 2007; Muranyi et al., 2002; Radsak et al., 1989; Reynolds et al., 2004; Scott & O'Hare, 2001). Notably, murine cytomegalovirus (MCMV) and, more recently, herpes simplex virus type 1 (HSV-1) have been reported to recruit cellular protein kinases C (PKCs) at the nuclear rim, and, as a consequence, induce an increase in phosphorylation of nuclear envelope proteins, including lamins and emerin, to promote nuclear lamina dissolution (Leach et al., 2007; Morris et al., 2007; Muranyi et al., 2002; Park & Baines, 2006). In addition, in HCMV, the virally encoded kinase pUL97 has been reported to be involved in this process by mediating the hyperphosphorylation of lamin A/C and p32, a component of the nuclear lamina that interacts with the lamin B receptor (Marschall et al., 2005). To some extent, this phenomenon resembles a physiological strategy employed by the cell during mitosis to disassemble the nuclear envelope (Broers & Ramaekers, 2004; Heald & McKeon, 1990; Peter et al., 1991; Stuurman, 1997; Thompson & Fields, 1996). In this context, two highly conserved herpesvirus gene products, homologous to HSV-1 pUL31 and pUL34 proteins, are considered to play a fundamental role in inducing host cell nuclear alterations. These two viral proteins physically interact, co-localize at the nuclear rim and are required for efficient virion release from the nuclear compartment, as shown from a number of studies of mutant viruses lacking these genes (Bubeck et al., 2004; Chang et al., 1997; Fuchs et al., 2002; Lotzerich et al., 2006; Reynolds et al., 2001; Roller et al., 2000; Shiba et al., 2000). In HCMV, the proteins homologous to HSV-1 pUL34 and pUL31 are named pUL50 and pUL53, respectively. We previously carried out a preliminary characterization of pUL53, and described this viral protein as a true-late tegumental component that co-localized with nuclear lamins at late stages of infection (Dal Monte et al., 2002). However, the roles of HCMV pUL53 and pUL50 during viral infection are still uncharacterized. In the present study, further analyses of HCMV pUL53 localization during infection are reported using a novel, highly specific antiserum to pUL53, together with the transient expression of this viral protein in association with its counterpart, pUL50. We also report that HCMV infection induces rearrangements of the nuclear lamina, and present data to support the view that pUL53 and pUL50 play a role in this process. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasmids.
Mammalian expression construct pc53FLAG has been described previously (Dal Monte et al., 2002). To express pUL53 in bacteria, the corresponding open reading frame (ORF) was amplified by PCR using appropriate primers and plasmid pc53FLAG as template. The PCR product was cloned into the linearized vector pCRT7/CT-TOPO (Invitrogen) to generate the bacterial expression vector pCRTOPO-UL53, encoding the UL53 ORF fused to C-terminal 6xHis and V5 epitopes. To generate pUL50-expressing clones, the UL50 ORF was amplified by PCR using appropriate primers and HCMV strain AD169 genome as template. The PCR product was inserted into pCRT7/CT-TOPO, producing the plasmid pCRTOPO-UL50. To obtain the eukaryotic expression vector pcDNA-UL50V5, pCRTOPO-UL50 was digested with the restriction enzymes XbaI and PmeI and the resulting fragment, containing the UL50 ORF in frame with the 6xHis and V5 epitopes, was subcloned in the EcoRV site of plasmid pcDNA3 (Invitrogen).
Expression and purification of pUL53–6xHis fusion protein.
The construct pCRT7TOPOUL53 was transformed into the Escherichia coli BL21(DE3) strain. For production of pUL53–6xHis, 10 ml fresh stationary-phase culture was inoculated into 500 ml Luria broth supplemented with ampicillin (50 µg ml–1) and was grown at 37 °C to an OD600 of 0.6. Protein expression was induced with 5 mM IPTG for 6 h at 28 °C with gentle shaking. The protein was purified by affinity chromatography using a ProBond Purification System for 6xHis-tagged proteins (Invitrogen), according to the manufacturer's instructions. The pUL53 affinity-purified protein was separated by SDS-PAGE and stained with Coomassie blue or transferred onto nitrocellulose membranes and detected by Western blotting (WB) with an antibody to the V5 epitope. WB analysis revealed two specific bands, one with a molecular mass of about 42 kDa and a second, more abundant species of 28 kDa, which was chosen for antibody production. The purified protein was separated by 9 % SDS-PAGE, stained with CuCl2, excised and electroeluted. After a desalting step in a PD-10 Desalting Column (Amersham Biosciences), the recombinant protein was concentrated by evaporation at 4 °C, using a UniVapo 150H (UniEquip), to a final concentration of 0.45 µg µl–1 and used to immunize laboratory mice.
Production of anti-pUL53 antibody.
To produce the polyclonal antiserum against pUL53, six BALB/c mice were immunized with the purified protein and three with PBS as negative controls. The immunogen (45 µg in 100 µl water) and the negative controls were prepared as an emulsion in an excess of Freund's complete adjuvant and injected once a week for 6 weeks into the intraperitoneal cavity of the BALB/c mice (Cevenini et al., 1991). The ascites fluid containing the polyclonal antibody (pAb) against pUL53 or the controls were harvested and tested to evaluate specificity by WB on mock and pc53FLAG-transfected COS7 cells (see Supplementary material S1 available in JGV Online). All immunization experiments were performed in accordance with the legal requirements and after approval of the Ethics Committee.
Transfection.
Subconfluent COS7 cells were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Transfected cells were fixed or harvested 24–48 h after transfection and analysed by indirect immunofluorescence (IIF) or immunoprecipitation (IP) and WB assay.
IIF.
Infected or transfected cells were fixed in 4 % paraformaldehyde in PBS for 10 min at 4 °C and permeabilized with methanol at –20 °C for 10 min. Cover slips were blocked with BSA (4 % in PBS) and incubated for 1 h at 37 °C with the following primary antibodies: mouse anti-FLAG M2 monoclonal antibody (mAb) (diluted 1 : 250; Sigma-Aldrich), mouse anti-V5 epitope mAb (1 : 500; Invitrogen), rabbit anti-V5 epitope pAb (1 : 80; Sigma-Aldrich), mouse anti-pUL53 pAb (1 : 100), goat anti-lamin A/C pAb (1 : 10; Santa Cruz Biotechnology), goat anti-lamin B pAb (1 : 10; Santa Cruz Biotechnology), and mouse IgG anti-gB mAb and mouse IgM 14-16 anti-gN mAb (both undiluted; kindly provided by M. Mach, University of Erlangen, Germany). After washes with PBS, cells were incubated for 1 h at room temperature with fluorochrome-conjugated secondary antibodies at the following dilutions: FITC-labelled chicken anti-mouse IgG and TRITC-labelled donkey anti-goat IgG (1 : 200; Santa Cruz Biotechnology) and Alexa Fluor 350-labelled goat anti-mouse IgM (1 : 1000; Invitrogen). Chromatin staining was performed using 4'6'-diamidino-2-phenylindole (DAPI; Invitrogen) diluted to 0.1 ng ml–1 for 3 min at room temperature. After washes with PBS, cover slips were mounted with polyvinyl alcohol mounting medium with DABCO (Sigma-Aldrich) and analysed using a fluorescence microscope (Eclipse 600l; Nikon) with a x100 oil-immersion objective. Digital images were acquired using Lucia Image software (Nikon).
IP.
COS7 cells were harvested 48 h post-transfection. Cells were washed with PBS and lysed in Triton-X lysis buffer [50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 % Triton-X, Complete Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science)] for 10 min on ice and sonicated at low intensity for 10 s. After clarification, supernatants were incubated with the appropriate antibody (4 µg per sample) overnight at 4 °C, with gentle rocking. The following day, 30 µl protein A/G beads (Santa Cruz Biotechnology) were added and incubated for 4 h. After washes with Triton-X lysis buffer, beads were resuspended in 2x Laemmli's buffer, boiled at 95 °C and centrifuged at 16 000 g for 5 min before analysing the supernatant by 10 % SDS-PAGE.
Subcellular fractionation.
Subcellular fractionation of infected and mock-infected HELFs was performed to separate cytoplasmic and nuclear proteins. Cells were resuspended in lysis buffer containing 10 mM Tris/HCl (pH 7.8), 1 % NP-40, 10 mM 2-mercaptoethanol and Complete Mini EDTA-free Protease Inhibitor Cocktail Tablets (Roche Applied Science). Separation was achieved by hypotonic shock and shearing; nuclei were pelleted by centrifugation at 300 g at 4 °C, and the supernatant, representing the cytoplasmic fraction, was clarified by centrifugation at 600 g. Pellets containing purified nuclei were resuspended in Triton-X lysis buffer, sonicated at low intensity and cleared from nuclear debris by centrifugation at 16 000 g at 4 °C for 10 min. After quantification using the Bradford assay (Bio-Rad) and addition of Laemmli's sample buffer, equal amounts of protein from nuclear and cytoplasmic extracts (50 µg) were analysed by 5–20 % gradient SDS-PAGE.
WB.
After separation by SDS-PAGE, proteins were transferred to Protran Nitrocellulose Transfer Membranes (Schleicher & Schuell Biosciences). Membranes were saturated with 5 % non-fat milk (Bio-Rad) in 0.1 % Tween 20 in PBS, washed with 0.1 % Tween 20 in PBS and probed with the following antibodies: mouse anti-pUL53 pAb (diluted 1 : 150); goat anti-lamin A/C pAb (1 : 100; Santa Cruz Biotechnology), goat anti-lamin B pAb (1 : 100; Santa Cruz Biotechnology), mouse anti-FLAG M2 mAb (1 : 1000; Sigma-Aldrich), mouse anti-V5 epitope mAb (1 : 5000; Invitrogen), goat anti-actin pAb (1 : 1000; Santa Cruz Biotechnology) or mouse anti-β tubulin mAb (1 : 200; Sigma-Aldrich). After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (diluted 1 : 10 000; Dako). Antibody binding was detected by ECL (Amersham Biosciences) using standard X-ray film (Kodak).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, panels i and ii, respectively). In contrast, at late stages of infection there were significant modifications to the nuclear shape, accompanied by about a threefold increase in nuclear size compared with uninfected cells (data not shown), as previously reported by Ruebner et al. (1965) and as similarly observed following HSV-1 infection in a process involving nuclear actin and the viral proteins pUL31 and pUL34 (Simpson-Holley et al., 2005). Importantly, marked alterations of the nuclear lamin labelling pattern were detected. Whereas at early stages of infection (48 h p.i.) no change was observed (data not shown), at later stages (96–120 h p.i.), in gB-positive cells, lamin A/C showed a ruffled staining pattern at the nuclear rim with the formation of invaginations, which increased in number and dimension during the progression of infection (Fig. 1b, panels i–iii); a similar pattern was observed for lamin B (Fig. 1b, panels iv–vi).
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, panel ii). A weak gN staining was detected in the cytoplasm of these cells (Fig. 2a, panel i). In the majority of infected cells, pUL53 formed aggregates on the nuclear rim (Fig. 2a, panel iv), previously described as ‘punctate patches’ (Dal Monte et al., 2002), only at later stages (96–120 h p.i.), suggesting that its localization is spatially and temporally determined as the infection proceeds. To confirm the predominantly nuclear localization of pUL53 and the specificity of the weak cytoplasmic signal, WB experiments were performed on nuclear and cytoplasmic extracts of mock- and virus-infected cells at the late stage of infection (96 h p.i.). As shown in Fig. 2(b), the analysis revealed a major species of about 42 kDa in the nuclear fractions of infected cells and a faintly detectable band in the cytosolic extracts. No specific signal was detectable in mock-infected samples. Cellular actin and β-tubulin staining was carried out to confirm equivalent protein loading and efficient subcellular fractionation, respectively, and showed comparable distribution in both infected and mock-infected extracts.
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, respectively, panels iv–vi). To assess whether the spatial reorganization of nuclear lamins was associated with changes in the amounts of these proteins being produced following infection, WB analysis was performed on cellular subfractions of infected and mock-infected cells. As shown in Fig. 4, two bands of 65 and 70 kDa corresponding to lamin A/C and one band of 68 kDa corresponding to lamin B were detected in infected and mock-infected nuclear fractions at equivalent levels, whilst no specific signal was detected in cytoplasmic extracts (data not shown), suggesting that no quantitative changes in the amounts of these nuclear proteins occurred following HCMV infection.
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, transiently expressed pUL53 localized within the nucleus with a diffuse pattern, in contrast to what was observed during virus infection. As several herpesvirus pUL53 homologues require the presence of their pUL50 counterparts for localization at the nuclear rim (Lake & Hutt-Fletcher, 2004; Muranyi et al., 2002; Reynolds et al., 2001; Yamauchi et al., 2001), we tested the effect of pUL50 on pUL53 localization, following transient co-expression of these two viral proteins. pUL50 is predicted to be a type II transmembrane protein with a long N-terminal cytoplasmic domain and a short C-terminal luminal tail. Consistent with this prediction, when expressed alone as a fusion protein with a V5 epitope, pUL50 localized mainly in the cytoplasmic compartment and perinuclear area, with a predominant endoplasmic reticulum staining pattern (Fig. 5d–f). However, when co-expressed with pUL53, both pUL53FLAG and pUL50V5 co-localized at the nuclear rim forming aggregates (Fig. 5g–i), in a pattern reminiscent of that observed for pUL53 in HCMV-infected cells. This observation indicated that pUL50 was sufficient to target pUL53 to the nuclear rim in the absence of other viral proteins. Their interaction was further investigated by IP experiments. Lysates of cells expressing either pc53FLAG or pcDNA50V5 alone and co-transfected cells were used to immunoprecipitate pUL53 with the anti-FLAG-tag antibody. As shown in Fig. 6, a specific band of about 42 kDa (Fig. 6a) corresponding to pUL53 was detected both in lysates expressing pUL53 alone and in lysates from cells co-expressing pUL50, whereas a band of about 43 kDa (Fig. 6b), corresponding to pUL50, was detected only in lysates of cells expressing both pUL50 and pUL53, indicating that the two proteins can be co-immunoprecipitated.
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). Unlike mock-transfected COS7 cells where both A- and B-type lamins localized in a smooth distribution at the nuclear periphery (Fig. 7a and b, panel ii), pUL53-expressing cells showed the occasional irregular distribution of lamin B together with areas of reduced staining and fenestrations (Fig. 7b, panel v). To a lesser extent, a similar pattern was detectable for lamin A/C (Fig. 7a, panel v). In contrast, transient expression of pUL50 induced the formation of cytoplasmic aggregates of lamin A/C and lamin B, which co-localized with pUL50, as shown in Fig. 7(a and b, panels vii–ix), respectively. These results indicated that, although both pUL50 and pUL53 are capable of promoting minor alterations of the nuclear lamina when expressed singly, these rearrangements are not comparable to those seen in the context of HCMV infection. We therefore investigated the effects of the pUL50–pUL53 complex on nuclear lamins in the absence of other viral products, by co-expressing the two viral proteins in COS7 cells and performing co-staining experiments. As pUL53 localized at the nuclear rim only after co-expression with pUL50, we considered pUL53 nuclear rim aggregates as a marker for pUL50–pUL53 complexes. Thus, experiments were performed to investigate the localization of nuclear lamins and pUL53 in cells expressing both pUL53 and pUL50. In cells expressing both proteins (see also Fig. 5a–c
), both lamin A/C and lamin B showed an irregular staining pattern, with formation of invaginations that co-localized with pUL50–pUL53 complexes, as shown in Fig. 7(a, panels x–xii) for lamin A/C and Fig. 7(b, panels x–xii) for lamin B. These observations showed that co-expression and aggregation of both pUL50 and pUL53 were sufficient to induce lamina alterations, closely resembling the staining patterns described during HCMV infection.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Farina et al., 2000; Lotzerich et al., 2006; Reynolds et al., 2001; Roller et al., 2000; Shiba et al., 2000; Ye & Roizman, 2000).
IIF analyses of nuclear lamins A/C and B, the major components of nuclear lamina, during HCMV infection showed that, at late stages of infection, both lamin A/C and B exhibited an irregular and ruffled distribution at the nuclear rim, with the formation of deep invaginations and herniations (see Fig. 1). Interestingly, such modifications closely resemble the infoldings at the inner nuclear membrane previously described by electron microscopy (Buser et al., 2007; Nassiri et al., 1998; Severi et al., 1988), which are proposed to be the preferential sites for virus budding at the nuclear envelope. Several studies on other members of the herpesvirus family claim that disruption of the nuclear lamina occurs in the presence of two highly conserved viral proteins, namely the homologues of pUL31 and pUL34 in HSV-1. The two viral products interact and co-localize at the nuclear boundary where they promote the egress of capsids from the nucleus by altering the structure of the host cell nucleus (Bjerke et al., 2003; Fuchs et al., 2002; Gonnella et al., 2005; Klupp et al., 2001; Muranyi et al., 2002; Reynolds et al., 2004, 2001). Therefore, we hypothesized that pUL53 and pUL50, the HCMV homologues of HSV-1 pUL31 and pUL34, respectively, might be similarly involved in inducing nuclear alterations. pUL53 has been identified by our group as a true-late protein component of the tegument that co-localizes nuclear lamins during the late phase of infection (Dal Monte et al., 2002). In this study, further analyses of the kinetics of pUL53 expression and localization were carried out. IIF and WB analyses showed that pUL53 localized almost completely at the nuclear rim of infected cells late in infection, with hardly any detectable cytoplasmic staining (Fig. 2a and b). Interestingly, in the early stages of infection (48 h p.i.), pUL53 formed small aggregates with a uniform distribution at the nuclear boundary (Fig. 2a, panels i–iii), whilst at later times (96–120 h p.i.), the homogeneous distribution at the nuclear rim was lost and pUL53 was localized in distinct punctate structures that decreased in number and increased in dimension (Fig. 2a, panels iv–vi). This phenomenon is not observed for the alphaherpesvirus homologues in HSV-1 and pseudorabies virus, or for the gammaherpesvirus Epstein–Barr virus (EBV), which are reported to spread stably on the nuclear surface in a smooth (Fuchs et al., 2002; Reynolds et al., 2001) or slightly punctate manner (Lake & Hutt-Fletcher, 2004), respectively. These observations suggest that pUL53 shares common features with its homologues in other herpesviruses, such as its kinetics of expression and localization at the nuclear rim, but also that pUL53 exhibits some differences in its localization following the course of infection by different herpesviruses. In this context, it is important to note that HSV-1 pUL31 only displays a punctate aggregation in the absence of the virally encoded kinase Us3, and that this has been described as a necessary viral factor for the correct localization of the UL31 product and its counterpart pUL34 during infection (Klupp et al., 2001; Reynolds et al., 2002). Interestingly, HCMV lacks a homologue of HSV-1 Us3, and it is reasonable to hypothesize that the diverse genomic content might determine differences in the localization pattern of pUL53. Subsequently, co-staining of pUL53 and nuclear lamins was performed to evaluate the potential involvement of this viral protein in rearrangement of the nuclear lamina (Fig. 3). At early stages of infection (48 h p.i.), pUL53 was uniformly distributed at the nuclear periphery and co-localized with unaltered nuclear lamins, whilst at later times (96–120 h p.i.), pUL53 aggregates were observed that strongly co-localized with lamin invaginations, suggesting a specific role for this protein in the remodelling of the nuclear rim. In addition, WB analyses of nuclear lamins in the presence of pUL53 indicated that the levels of these cellular proteins were not affected by HCMV infection (Fig. 4), in agreement with previous reports (Marschall et al., 2005; Radsak et al., 1989). However, unlike the findings for the EBV homologues (Gonnella et al., 2005), our IP experiments did not show co-immunoprecipitation of pUL53 with either lamin A/C or lamin B (data not shown), suggesting that, in HCMV, the role of pUL53 might be indirect and it could be required for the recruitment of other viral or cellular factors directly responsible for disruption of the nuclear lamina.
When expressed in the absence of other viral proteins, pUL53 failed to aggregate at the nuclear rim and localized diffusely in the nucleus (Fig. 5a; see also Dal Monte et al., 2002) as previously reported for other herpesvirus pUL53 homologues (Lake & Hutt-Fletcher, 2004; Muranyi et al., 2002; Reynolds et al., 2001; Ye & Roizman, 2000), suggesting that other factors are required for its correct distribution during infection. Importantly, nuclear rim localization of pUL53 could be restored following co-expression with pUL50, the homologue of HSV-1 pUL34 (see Fig. 5). The interaction between the viral products was further demonstrated by co-immunoprecipitation (Fig. 6). These results suggested that pUL50 is necessary and sufficient to induce the distribution of pUL53 into large aggregates at the nuclear rim during HCMV infection.
Similar experiments have been reported for the corresponding proteins of MCMV, M50 and M53 (Muranyi et al., 2002). Taken together, these data suggest strong homology not only in terms of amino acid sequence but also in functional terms between the pUL50 and pUL53 homologues of these two members of the subfamily Betaherpesvirinae. This hypothesis is also supported by recent findings, where M50 or M53 of MCMV was replaced with their respective HCMV homologue in recombinant MCMVs in which the functional properties of the complex were maintained (Schnee et al., 2006). However, characteristic localization patterns and evidence for an interaction have also been described for other herpesvirus homologues of pUL50 and pUL53, suggesting that their roles are highly conserved not only among members of the subfamily Betaherpesvirinae, but throughout all herpesviruses (Fuchs et al., 2002; Lake & Hutt-Fletcher, 2004; Reynolds et al., 2001; Yamauchi et al., 2001).
Interestingly, after co-expression of pUL50 and pUL53, the staining pattern observed for nuclear lamins was identical to that reported during HCMV infection: pUL50–pUL53 aggregation at the nuclear rim induced alterations and invaginations of both lamin A/C and lamin B, as seen in HCMV-infected cells (Fig. 7a and b). However, pUL50 and pUL53 were also able to induce occasional nuclear lamin impairment when expressed alone: pUL50 was found at the nuclear membrane, but also in the cytoplasm, co-localizing with aberrant cytosolic lamin A/C and B, whilst pUL53 expression caused fenestrations and thinning of nuclear lamins. Taken together, the current data suggest that nuclear lamins undergo remarkable conformational and structural alterations in limited areas at the nuclear rim, corresponding to the localization sites of pUL53–pUL50 complexes, both during infection and when the two proteins are co-expressed in the absence of virus infection.
Similar results have been reported by Muranyi et al. (2002) after co-expression of MCMV M50 and M53 and following MCMV infection, strengthening the hypothesis that the MCMV and HCMV pUL50–pUL53 complexes share a common function at the nuclear rim. It has also been suggested that M50 is responsible for the direct modification of the nuclear lamina, whilst M53 modulates its effects. With HCMV, we were not able to detect any exclusion of nuclear lamins from areas of pUL50 accumulation at the nuclear envelope following transient expression, but only occasional relocalization in the cytoplasm, whilst lamina fenestrations and interruptions were observed sporadically after overexpression of pUL53. Dramatic changes in the distribution of nuclear lamins were only detectable in the presence of both pUL50 and pUL53, suggesting that both viral proteins contribute to this process.
Previously, it was reported that, during MCMV infection, the homologous proteins M50 and M53 recruit and co-localize at the nuclear rim with aggregates of cellular calcium-dependent PKCs (Muranyi et al., 2002). For HCMV, it has been reported that infection induces hyperphosphorylation of nuclear lamins (Muranyi et al., 2002) and that this is mediated by the HCMV-encoded kinase pUL97 (Marschall et al., 2005). In this context, one hypothesis could be that pUL50–pUL53 complexes induce structural alterations of nuclear lamins, in parallel with other cellular or viral factors responsible for the biochemical modifications (e.g. cellular PKCs or HCMV pUL97) or that they act as a scaffold to retain these factors at the sites of nuclear lamina damage. However, further studies are required to understand the possible interactions of pUL50 and pUL53 with other elements involved in HCMV infection.
In conclusion, the observations that (i) invaginations of nuclear lamins strongly resemble the infoldings at the INM where virion budding preferentially occurs (Buser et al., 2007; Nassiri et al., 1998; Severi et al., 1988); (ii) pUL53 co-localizes with nuclear lamin alterations during HCMV infection; (iii) pUL50 is sufficient to induce pUL53 localization in aggregates at the nuclear rim; and (iv) the pUL50–pUL53 complex is sufficient to cause and co-localize with lamina invaginations upon transient expression as seen in infected cells, strongly suggest that the two viral proteins play a critical role in the production of viral progeny, by creating favourable conditions for viral egress from the nucleus to the cytoplasm.
Whilst this article was being reviewed, a related article reporting the association of HCMV pUL50 and pUL53 with the nuclear lamina was published (Milbradt et al., 2007).
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ACKNOWLEDGEMENTS |
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Received 14 August 2007;
accepted 16 November 2007.
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