Virus Research 175 (2013) 143– 150

Contents lists available at SciVerse ScienceDirect

Virus Research

jo ur nal home p age: www.elsev ier .com/ locate /v i rusres

Short communication

KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and altersemerin distribution

Antonella Farinaa,!, Roberta Santarelli a, Rossella Bloisea, Roberta Gonnellaa, Marisa Granatoa,Roberto Beib, Andrea Modestib, Mara Cironea, Luiza Bengtssonc,Antonio Angelonia, Alberto Faggionia

a Istituto Pasteur Fondazione Cenci Bolognetti, Dipartimento di Medicina Sperimentale, Sapienza Università di Roma, Italyb Department of Clinical Sciences and Translational Medicine, Faculty of Medicine, University of “Tor Vergata” Roma, Italyc Leibniz-Institut für Molekulare Pharmakologie (FMP) and Max-Delbrück-Centrum für Molekulare Medizin (MDC), D-13125 Berlin, Germany

a r t i c l e i n f o

Article history:Received 17 December 2012Received in revised form 28 March 2013Accepted 1 April 2013Available online 23 April 2013

Keywords:KSHVORF67ORF69Viral egressNuclear envelope

a b s t r a c t

p29, a newly identified Kaposi’s sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67,the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other her-pesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Uponchemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33,encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both pro-teins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cellsshowed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereasother nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalizationof emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerindestabilization might represent the first step in nuclear lamina disassembling, a process necessary fornucleocapsid maturation.

© 2013 Elsevier B.V. All rights reserved.

Kaposis’s sarcoma associated herpesvirus (KSHV), also knownas human herpesvirus 8 (HHV-8), is a lymphotropic virus detectedin biopsies of all clinical and epidemiological forms of Kaposi’ssarcoma (KS) and linked to two lymphoproliferative disorders,such as primary effusion lymphomas (PEL) and multicentric Castle-man’s disease (MCD) (Ganem, 2007). Like all herpesviruses, KSHVis able to perform latent and lytic infection. Although in tumorcells the infection is predominately latent, recent data pointedout the importance of lytic replication in viral oncogenesis (Zhaoet al., 2007). During herpesvirus assembly, DNA replication, tran-scription, capsid formation and viral DNA packaging occur in thenucleus. Then capsids leave the nucleus and gain access to thecytoplasmic compartment (primary egress) passing through thenuclear lamina (Granzow et al., 2001; Morrison and Delassus,2011). This cellular structure is a dense fibrillar network inside thenucleus of eukaryotic cells, composed primarily of type V inter-mediate filament proteins (lamin A, lamin B and lamin A/C) andseveral transmembrane proteins that bind to lamins and may be

! Corresponding author at: Dipartimento di Medicina Sperimentale, Viale ReginaElena 324, 00161 Roma, Italy. Tel.: +39 06 4461500; fax: +39 06 4456229.

E-mail addresses: alberto.faggioni@uniroma1.it, antoeffe2009@yahoo.it(A. Farina).

tightly associated with the nuclear lamina (Cibulka et al., 2012;Gruenbaum et al., 2005; Senior and Gerace, 1988). Besides provid-ing mechanical support, the nuclear lamina regulates importantcellular events such as cellular DNA replication and cell division(Manilal et al., 1998). Many functions have been attributed to thenuclear lamina-related proteins such as a role in chromatin orga-nization, transcription, DNA synthesis, cellular differentiation, cellmigration, signal transduction and progression of the cell cycle(Gruenbaum et al., 2005). Among lamin associated proteins themost important are emerin (a type II integral membrane pro-tein), whose function is to mediate membrane anchorage to thecytoskeleton (Bengtsson and Wilson, 2004), lamin B-receptor (LBR),that provides essential chromatin docking sites at the nuclear enve-lope (Pyrpasopoulou, 1996) and LUMA, that interacts with emerinand influences its distribution (Bengtsson and Otto, 2008). Defectsin the genes encoding for nuclear lamin (such as lamin A and laminB1), or for other proteins associated to the nuclear lamina, havebeen implicated in a variety of diseases known as laminopathies(Mendez-Lopez and Worman, 2012; Worman, 2012). It has beenpreviously reported that during herpesvirus maturation two highlyconserved proteins (UL31–UL34 in HSV-1 and PRV, BFRF1–BFLF2 inEBV and UL50–UL53 in HuCMV) (Camozzi et al., 2008; Farina et al.,2005, 2000; Fuchs et al., 2002; Gonnella et al., 2005; Granzow et al.,2001; Lotzerich et al., 2006; Milbradt et al., 2009) have a key role

0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.virusres.2013.04.001

144 A. Farina et al. / Virus Research 175 (2013) 143– 150

in primary egress. Indeed, these proteins form a nuclear egresscomplex (NEC) that interacts directly with the nuclear laminsand the nuclear lamina-associated proteins, disassembling lamina’snetwork and enabling nucleocapsids to pass through the nuclearmembrane. While this mechanism has been described for HSV-1,CMV and EBV (Gonnella et al., 2005; Granzow et al., 2001), littleis known about the maturation of KSHV. We previously identifiedand characterized the product encoded by ORF69, the positionalhomologue of HSV-1 UL31, a 33 kDa a lytic protein strongly associ-ated to the nuclear matrix, which localizes in the nucleus of TPAinduced PEL cells (Santarelli et al., 2008). More recently, Desaiet al. (2012) confirmed and extended our results studying KSHVnuclear egress complex using a baculovirus expression system andshowed a direct interaction between ORF67 and ORF69 encodedproteins, homologues to UL34/BFRF1 and UL31/BFLF2, respectively.The authors also described a role of this viral protein complex inthe induction of nuclear membrane reduplication and productionof vesicles similar to herpesvirus virions, in insect cells. Thus, theaim of our present work was to study the ORF67 expression and

localization in PEL cell lines, to examine its interaction with ORF69encoded p33 and to investigate whether these proteins interactwith nuclear membrane and/or nuclear matrix proteins. To pro-duce His-ORF67 fusion protein for mice immunization, the 843 bpRT-PCR fragment containing ORF67, obtained from BC3 after 48 htreatment with TPA (20 ng/ml), has been cloned into pTRC-HisAvector (primers used were JV-U 5!-CCA GCC CCA CCT CGA GGTCGC CAT GAG TGT-3!; JV-D 5!-ACA CGC GGT GGTA CCT CAG CTGGGC CTC AT-3!). As shown in Fig. 1A, the selected A7 monoclonalantibody specifically recognized a protein of approximately 29 kDa(p29) in Western blot analysis in BC3 and BCBL-1 PEL cell linestreated with TPA to induce KSHV lytic cycle, as well as in pCMV-ORF67 transiently transfected HEK293 cells. p29 expression wasnot detected in untreated BC3 and BCBL-1 and no cross reactiv-ity with BFRF1 protein (p29 homologue in EBV) was observed inTPA/NaButyrate treated B95-8 EBV-producing cell line. We theninvestigated the cellular localization of p29. To this end, IFA exper-iments were performed on BC3 cells treated with TPA using A7monoclonal antibody. Fig. 1B (left panel) shows that p29 is mainly

Fig. 1. Expression and localization of KSHV p29. (A) Expression of ORF67 encoded protein. A7 MoAb recognized a 29 kDa protein in TPA induced BC3 and BCBL1 as well in 293ORF67 transfected cells by Western blot analysis. No signal was detected in uninduced cells. No cross-reactivity with EBV BFRF1 protein was seen in induced (T/B) B95-8 cells.(B) p29 cellular localization. IFA analysis performed on TPA treated BC3 cells shows a perinuclear distribution of p29 (green upper panel), while the ectopic expression of theprotein in 293 cells (right panel) shows that the protein is mainly localized on the nuclear rim. Nuclei were counterstained with DAPI. Bars = 10 !m. (C) Induced BC3 cellswere fractionated as described and nuclear (N), cytoplasm (C), membrane (M) and cytoskeletal (Ck) fractions were analyzed by Western blot analysis, revealing the presenceof p29 both in nuclear and in cytoskeletal fraction. (D) p29 is associated to the nuclear matrix. Nuclear matrix fractions, were separated by SDS-PAGE and immunoblottedusing A7 MoAb: p29 is present in both soluble (I) and in the nuclear matrix fractions (IV) of induced BC3 cells. ":p53 has been used to monitor the quality of nuclear matrixfractions. (E) p29 is expressed “in vivo”: p29 positive (1–2) and p29 negative (3) KSHV seropositive donors from Sardinia and sera from AIDS-associated KS patients (4) werescreened by Western blot toward ptrcA lisate (A) or purified His-p29 fusion protein (A67). Anti-p29 A7 monoclonal antibody has been used as a control. (For interpretationof the references to color in figure legend, the reader is referred to the web version of the article.)

A. Farina et al. / Virus Research 175 (2013) 143– 150 145

Table 1Summary of p29 seroprevalence in individuals infected with KSHV and healthydonors.

No. of cases No. of p29 seropositive %

AIDS KS 17 14 82.3KSHV seropositive donors

from Sardinia57 13 22.8

Healthy donors 36 1 2.7

localized on the nuclear membrane and is distributed in patchesduring KSHV replication. A similar distribution was observed alsoafter ectopic expression of p29 in HEK293 cells (Fig. 1B right panel),thus indicating that its localization on the nuclear membrane doesnot depend on the presence of other viral proteins, similarly toEBV BFRF1 (Farina et al., 2004). Furthermore, Western blot anal-ysis performed on subcellular fractions of TPA-treated BC3 cellsconfirmed that p29 is present in the nuclear extract, although alow amount of the protein is also detectable in the cytoskeletalfraction (Fig. 1C). The same distribution was observed in TPA-treated BCBL1 cell line (data not shown). We then asked if thisprotein was associated with the nuclear matrix and to this endwe used a specific nuclear fractionation protocol (Santarelli et al.,2008). Briefly, in the first step non ionic detergent lysis allowedsoluble proteins extraction (fraction I). In the next step, to precip-itate chromatin-associated proteins, chromatin was solubilized byRNase-free DNase I digestion and ammonium sulfate was added(fraction II). Finally, the pellet was washed with 2 M NaCl (frac-tion III) and the remaining pellet, containing the structural nuclearmatrix and the nuclear matrix-associated proteins, was solubi-lized in 8 M urea (fraction IV). Fractions were then analyzed byimmunoblotting. Fig. 1D shows that p29 is detectable in both frac-tion I and IV (upper panel), thus indicating that a large amount ofthis protein is also associated to the nuclear matrix. Furthermore,in order to control collected fractions we used an anti-p53 antibody(lower panel).

Since previous studies demonstrated that antibodies directedtoward EBV BFRF1 protein were found in the sera of BL and NPCpatients and that this protein is a good marker of active viral repli-cation (Farina et al., 2000), we investigated if antibodies againstp29 were present in sera of AIDS-associated KS, KSHV seroposi-tive subjects from Sardinia without KS (Santarelli et al., 2001), thatrepresents an intermediate seroprevalence from an high risk areafor classic KS, and healthy donors. As shown in Table 1 we foundantibodies to p29 in 82% sera of AIDS-associated KS patients and in22.8% of KSHV seropositive subjects, as assessed by Western blotanalysis (Fig. 1E). The results of the screening were also confirmedby the reactivity of sera by IFA on 293HEK cells transfected withCMV-ORF67 and by testing sera on purified p29 protein coatedELISA plates (data not shown). Thus, we concluded that this proteinstimulates a humoral response, mainly in KS patients.

As stated above, we previously identified and characterized thep33 protein, encoded by KSHV ORF69 and homologue to HSV-1UL31 and EBV BFLF2 (Santarelli et al., 2008). When expressed in293HEK cells p33 appears diffused in the nuclei, while it is local-ized at the nuclear rim when cotransfected with GFP-ORF67. Inaddition, these two proteins colocalized following cotransfectionin 293HEK cells. In order to evaluate whether p29 and p33 alsocolocalize in PEL cell lines, we performed a double IFA stainingin TPA-treated BC3 cells (all images have been analyzed using40! ApoTome mycroscope Zeiss, Germany). To this end, p29 wasstained with A7 moAb (red) and p33 with the pp27 rabbit poly-clonal antibody (green) (Fig. 2A). Merged images revealed that thetwo proteins colocalize on the nuclear rim during KSHV replication.As a control, nuclei were stained with DAPI. These results indi-cate that p29 and p33 have a behavior similar to their homolog

proteins in HSV-1/2, EBV, CMV, mCMV and PrV (Fuchs et al., 2002).We next examined whether during KSHV lytic cycle the nuclearmembrane and/or nuclear lamina, which normally represent a bar-rier for herpesvirus nuclear egress, were modified. In particular weanalyzed the distribution of the following cellular proteins: laminB1 (LB1), LBR, LUMA and emerin (Gruenbaum et al., 2005; Mendez-Lopez and Worman, 2012). To this aim we performed double IFAanalysis on BC3 cells treated with TPA, using polyclonal antibod-ies directed against the nuclear cellular proteins and monoclonalanti-p29 or -p33 antibodies. As shown in Fig. 2B–D, LB1, LBR, andLUMA localization was not modified during KSHV replication. Con-versely, we observed a different distribution of emerin in the cellswhere KSHV lytic cycle was activated and consequently p29, p33and K8.1 were expressed (Fig. 2F, G and H, respectively). Similarresults were obtained in TRExBCBL-1-Rta cells induced by deoxicy-cline treatment (1 mg/ml) (data not shown) (Nakamura et al., 2003),indicating that this effect is directly correlated to viral replicationand does not depend on the chemical treatment used to induceKHSV replication. As a control in Fig. 2E we report the immunofluo-rescence assay performed with anti-emerin polyclonal antibody onKSHV latently infected BC3 cell: in these cells, emerin is normallydistributed on the nuclear membrane. We then analyzed the effectof the ectopic expression on the above mentioned nuclear proteinin HEK293 cells. As shown in Fig. 3A–C, IFA analysis indicated thatthe expression of p29 in these cells did not affect distribution of LB1,LBR or LUMA, while emerin was delocalized and confined in peri-nuclear dots (Fig. 3D). Conversely, single transfection of ORF69 inHEK293 cells did not affect LUMA, LB1, LBR (not shown) or emerinlocalization (Fig. 3E). As a control we show emerin distribution inuntransfected HEK293 cells (Fig. 3F) and in HEK293 transfectedwith the pHD1013 control plasmid (Fig. 3G). These data stronglyindicate that p29 expression, besides its role to target p33 at thenuclear rim, is sufficient to dislocate the emerin, whose function isto stabilize nuclear lamins and suggest that, like other herpesvirusfamily homologues, p29 is a multifunctional protein. As alreadydescribed, p33 reaches the nuclear rim only in presence of p29.In 293HEK cells cotransfected with ORF67/ORF69 double stainingwith H8 monoclonal antibody (green) and anti-emerin polyclonalantibody (red) reveals that co-expression of p29 and p33 furtherenhances the emerin mislocalization, in comparison to p29 singletransfection (Fig. 4A). However LBR, Lamin B1 and LUMA remainproperly localized (Fig. 4B–D), suggesting that p29–p33 complex isaffecting only emerin distribution.

During HSV-1 replication it has been described that UL34recruits US3 viral kinase and PKC-delta, inducing hyperphos-phorylation of emerin and altering lamins. Moreover, it is welldocumented that a second viral kinase, UL13, phosphorylates US3in infected cells and regulates UL34 and UL31 localization (Katoet al., 2006; Leach and Roller, 2010; Mou et al., 2009). Otherwise,gamma herpesviruses show a different behavior: during EBV lyticreplication, the viral kinase BGLF4, homolog to UL13, targets thenucleus through interaction with nucleoporin and induces disas-sembly of the nuclear lamina (Lee et al., 2008). In KSHV a viralkinase, known as ORF36, homologue to BGLF4, has been identified(Hamza et al., 2004), but nothing is known regarding its interac-tion with other viral proteins or nuclear lamins nor its role in viralegress. Using an ORF36 expression plasmid and polyclonal antibod-ies directed against ORF36 (Hamza et al., 2004) we did not observecolocalization with ORF67 or ORF69 in preliminary experiments(data not shown).

Finally, to investigate how KSHV alters lamina and nuclearmembrane proteins during lytic replication we performed West-ern blotting analysis on BC3 cells (Fig. 5A). For this purpose KSHVlytic cycle was induced with TPA for 24, 48 and 72 h and percent-age of induced cells was monitored by IFA (30–40% of induction)prior to lyse cells. Total cellular lysates as well as soluble (S) and

146 A. Farina et al. / Virus Research 175 (2013) 143– 150

Fig. 2. Localization of viral and nuclear proteins during KSHV replication in BC3 cells. (A) During viral replication, KSHV p33 and p29 proteins colocalized on the nuclear rimas shown by double IFA analysis performed using rabbit anti-p33 (green) and A7 MoAb (red); merged image shows that both proteins are expressed in patches. Followinginduction of KSHV replication in BC3 cells, IFA analysis was performed also on components of the nuclear membrane. Specific antibodies directed against LB1, LBR and LUMAwere stained with Cy3-conjugated secondary antibodies (respectively (B), (C) and (D)), while A7 Moab specific to p29 was stained with a FITC conjugated Ab. Merged imagesshow that the above mentioned cellular proteins are properly distributed during replication. Conversely, double staining performed on induced BC3 cells with anti-emerinand anti-p29, p33 and K8.1 antibodies showed an altered distribution of the emerin ((F), (G) and (H), respectively). This protein is enclosed in dots inside the nucleus and nolonger distributed on the nuclear rim as in not induced cells (E). Bars = 10 !m. (For interpretation of the references to color in figure legend, the reader is referred to the webversion of the article.)

A. Farina et al. / Virus Research 175 (2013) 143– 150 147

Fig. 3. Ectopic expression of p29 is able to alter emerin distribution. To analyze the effect of p29 on nuclear membrane, double IFA staining was performed on HEK293transiently transfected cells with p29 expression plasmid. Ectopic expression of KSHV p29 (green), does not interfere with the localization of cellular protein such as LB1,LBR and LUMA (red) (respectively (A), (B) and (C)), as shown by double IFA. Conversely, transient expression of p29 (green) alters emerin distribution: this protein is nolonger homogeneously distributed on the nuclear rim but is organized in dots (D). Ectopic expression of p33 alone is not able to alter emerin distribution (E). As a control weshow emerin distribution in untransfected or transiently transfected with the plasmid control pHD1013 Hek293 cells ((F) and (G), respectively): emerine is homogeneouslydistributed on the nuclear rim (green). Bar = 10 !m. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)

unsoluble (P) protein fractions were isolated (Morris et al., 2007)and immunoblotted. As shown in Fig. 5A, we did not observe post-translational modifications of LB1, LUMA and emerin during thefirst 48 h after induction of lytic cycle either in total lysate or insoluble/insoluble fractions. Otherwise, LBR appeared reduced after

48 hr induction in total lysate as in soluble/insoluble fractions. Inthe late stages of KSHV replication (72 h), the amount of all exam-ined nuclear proteins is decreased, probably due to the activation ofapoptosis (Kramer et al., 2008). Since there is not a useful markerpresents in equal amount of both soluble and pellet fraction we

148 A. Farina et al. / Virus Research 175 (2013) 143– 150

Fig. 4. Ectopic co-expression of p29 and p33 in HEK293 cells dramatically enhances emerin distribution. (A) 293 cells were transiently co-transfected with ORF69 and ORF67expression plasmids. 48 h post transfection, cells were stained with H8 monoclonal antibody (green) and with rabbit anti-emerin (red). Double IFA staining shows that whenboth proteins are co-expressed p33 is localized on the nuclear rim and emerin is redistributed in dot-like structures and in some of them colocalyzes with p33 (merge). Asa control, cotransfected cells were stained with specific antibodies directed against LBR, LB1 and LUMA: double IFA staining showed that these proteins are not affected bythe simultaneous expression of KSHV proteins ((B), (C) and (D), respectively). (For interpretation of the references to color in figure legend, the reader is referred to the webversion of the article.)

performed Coomassie staining of the membrane as loading con-trol, as in Morris et al. (2007). Same experiments were performedon TRExBCBL-1-Rta cell line (not shown).

As previously shown, IFA experiments demonstrated thatp29 and p33 affect emerin localization, hence we evaluatedwhether ectopic expression of these proteins induced emerinpost-translational modifications. To this aim we analyzed solubleand insoluble fractions of 293HEK cells transfected with ORF67alone or in combination with ORF69. As shown in Fig. 5B, weobserved new isoforms of emerin in the insoluble fraction of ORF67and ORF67/ORF69 transfected cells. These products were stronglyreduced after 1hr treatment with alkaline phosphatase at 37 !C(lane +), thus indicating that emerine is indeed phosphorylated byexpression of KSHV p29. Moreover, in order to test the integrityof the nuclear lamina, we performed immunoblot with anti-LaminB1 antibody. As shown in Fig. 5B (upper panel), after transfectionof ORF67 we observed Lamin B1 degradation products with a dif-ferent molecular weight, that are strongly reduced after alkalinephosphatase treatment (lane +). Lamin B1 fragments are enhancedin ORF67–ORF69 cotransfected cells. Otherwise, transfection ofpHD1013 plasmid control did not induce lamin B degradation.No alterations of LUMA or LBR were observed (data not shown).Thus, these results strongly support the evidence that both pro-teins are involved in nuclear lamin destabilization process, a criticalstep during herpesvirus nucleocapsids egress. In addition, to betteraddress whether the qualitative evidence of emerin mislocaliza-tion could have a relationship with it’s association to a specificnuclear or cellular compartment, we performed nuclear matrix andcellular fractionation experiments. Fig. 5C shows nuclear matrix

fractionation of TPA treated or untreated BC3 cells analyzed withanti-emerin antibody. Here we observed that small amount ofemerin was present in soluble fraction of TPA treated BC3 cells(fraction I lane T), whereas was not present in untreated cells (lane"); however, most of the protein remain tightly anchored to thenuclear matrix also during viral replication (fraction IV lane +), thusindicating that the binding with this structure is stable (Squarzoniet al., 1998). As control of collected fraction we show anti-p53immunoblot. Finally, in order to test whether emerin is reorga-nized through cellular compartments during lytic replication, wealso performed a partial cell fractionation of BC3 TPA treated oruntreated cells. As shown in Fig. 5D, we did not observe signifi-cant alteration of emerin amount in nuclear extract of TPA treatedcompared to latently infected cells. However we did not detect thisprotein in cytoslic fraction of TPA induced BC3, while we foundemerin in membrane/cytoskeletal fraction. To monitor the qualityof the cellular fraction we show the !-actin immunoblot. Takentogether these data indicate that during KSHV lytic cycle emerinis slightly redistributed in cell and nuclear matrix compartmentsand not degraded, a behavior similar to that observed during celldivision (Vaughan et al., 2001).

In conclusion, these data add new information in KSHV biology:p29 is a nuclear membrane protein expressed during viral repli-cation and strongly antigenic in vivo, as anti-p29 antibodies weredetected in sera of AIDS-related KS and KSHV-seropositive donors.ORF67 and ORF69 products colocalize at the nuclear membrane inPEL cell lines during KSHV lytic cycle and p29 expression is able toalter the distribution of emerin, that has a pivotal role in maintain-ing the correct architecture of the inner nuclear membrane (IM).

A. Farina et al. / Virus Research 175 (2013) 143– 150 149

Fig. 5. Analysis of biochemical alterations of nuclear envelope (NE) proteins. (A) Crude lysate (T) as well as soluble (S) and insoluble fractions (P) of TPA induced BC3 cellswere separated on 4–12% Novex gel and immunoblotted to check alterations of nuclear envelope proteins. Within 72 h post induction, no significant alterations were seenin LBR, LB1, LUMA or emerin. Coomassie staining is reported as loading control. (B) Fractionation protocol has been performed also on 293Hek cells transfected with thepHD1013 control plasmid, ORF67 alone or in combination with ORF69. Insoluble fractions (P) have been treated with alkaline phosphatase (+) or untreated (!), resolved on15% SDS-PAGE and analyzed by Western blot. Upper panel shows Lamin B1 immunoblot: faster migrating bands are lamina’s degradation products induced by transfectionof ORF67 plasmids alone or in combination with ORF69. Transfection with ORF67 or with ORF67/ORF69 plasmids (middle panel) induce hyperphosphorylations of emerin ininsoluble fraction. As a loading control we report Commassie staining of the membrane. (C) Nuclear matrix fractionation of TPA treated (lane T) or untreated BC3 cells (lane!). Immunoblot performed with anti-emerin antibody shows that emerin is mainly associated to the nuclear matrix (fraction IV) although a small amount is detectable insoluble fraction (I) of TPA treated cells. (D) BC3 cells were fractionated in nuclear (N), cytosol (C) and membrane/cytoskeletal (M/Ck) fractions. Lane 1 represent untreatedcells; lane 2 are TPA induced cells. Emerin amount is comparable in nuclear fraction whereas is enriched in membrane/cytoskeletal fraction of TPA treated BC3 cells andcompletely undetectable in cytosol. To monitor the quality of the fractionation assay we performed an immunoblot against !-actin.

Furthermore p29 induces the appearance of novel emerin phospho-species. The combined expression of p29 and p33 robustly increasesthe mislocalization of the emerin, but does not alter its phosphor-ylation state, indicating that probably p33 softens the functionof p29 in order to obtain a fine modulation of the physiologicaleffect. In addition, lamin B1 appeared disrupted following the sin-gle or combined expression of both viral proteins. Taken togetherthese data suggest that emerin destabilization, induced during viralreplication through p29, and lamin B1 disruption promoted byp29–p33 co-expression represent the first step in nuclear laminadisassembly, a mechanism necessary for nucleocapsid maturation.Moreover, our data suggest a possible role of KSHV in the alterationof cellular proteins observed in laminopathies, diseases charac-terized by an altered function of lamins and lamins-associatedproteins.

Acknowledgments

This work was partially supported by grants from Associa-tion Italiana per la Ricerca sul Cancro (AIRC) and from Ministerodell’Istruzione, dell’Università e della Ricerca (MIUR). We thankProf. J.U. Jung for the generous gift TRExBCBL-1-Rta cells and Dr.Y. Izumiya and Prof. P.A. Luciw for kindly provide FLAG-v Kinaseexpression plasmid and related antibody.

References

Bengtsson, L., Otto, H., 2008. LUMA interacts with emerin and influences its dis-tribution at the inner nuclear membrane. Journal of Cell Science 121 (Pt 4),536–548.

Bengtsson, L., Wilson, K.L., 2004. Multiple and surprising new functions for emerin,a nuclear membrane protein. Current Opinion in Cell Biology 16 (1), 73–79.

150 A. Farina et al. / Virus Research 175 (2013) 143– 150

Camozzi, D., Pignatelli, S., Valvo, C., Lattanzi, G., Capanni, C., Dal Monte, P., Landini,M.P., 2008. Remodelling of the nuclear lamina during human cytomegalovirusinfection: role of the viral proteins pUL50 and pUL53. Journal of General Virology89 (Pt 3), 731–740.

Cibulka, J., Fraiberk, M., Forstova, J., 2012. Nuclear actin and lamins in viral infections.Viruses 4 (3), 325–347.

Desai, P.J., Pryce, E.N., Henson, B.W., Luitweiler, E.M., Cothran, J., 2012. Reconstitu-tion of the Kaposi’s sarcoma-associated herpesvirus nuclear egress complex andformation of nuclear membrane vesicles by coexpression of ORF67 and ORF69gene products. Journal of Virology 86 (1), 594–598.

Farina, A., Santarelli, R., Gonnella, R., Bei, R., Muraro, R., Cardinali, G., Uccini, S., Rag-ona, G., Frati, L., Faggioni, A., Angeloni, A., 2000. The BFRF1 gene of Epstein–Barrvirus encodes a novel protein. Journal of Virology 74 (7), 3235–3244.

Farina, A., Cardinali, G., Santarelli, R., Gonnella, R., Webster-Cyriaque, J., Bei, R.,Muraro, R., Frati, L., Angeloni, A., Torrisi, M.R., Faggioni, A., 2004. Intracellu-lar localization of the Epstein–Barr virus BFRF1 gene product in lymphoid celllines and oral hairy leukoplakia lesions. Journal of Medical Virology 72 (1),102–111.

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 isessential for efficient primary viral envelopment and egress. Journal of Virology79 (6), 3703–3712.

Fuchs, W., Klupp, B.G., Granzow, H., Osterrieder, N., Mettenleiter, T.C., 2002. Theinteracting UL31 and UL34 gene products of Pseudorabies virus are involvedin egress from the host-cell nucleus and represent components of primaryenveloped but not mature virions. Journal of Virology 76 (1), 364–378.

Ganem, D., 2007. In: Fields Virology (Ed.), Kaposi’s Sarcoma-associated Herpesvirus., 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA, p. 13.

Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., Granato, M.,Modesti, A., Frati, L., Delecluse, H.J., Torrisi, M.R., Angeloni, A., Faggioni, A., 2005.Characterization and intracellular localization of the Epstein–Barr virus proteinBFLF2: interactions with BFRF1 and with the nuclear lamina. Journal of Virology79 (6), 3713–3727.

Granzow, H., Klupp, B.G., Fuchs, W., Veits, J., Osterrieder, N., Mettenleiter, T.C., 2001.Egress of alphaherpesviruses: comparative ultrastructural study. Journal of

Virology 75 (8), 3675–3684.Gruenbaum, Y., Margalit, A., Goldman, R.D., Shumaker, D.K., Wilson, K.L., 2005.

The nuclear lamina comes of age. Nature Reviews. Molecular Cell Biology 6 (1),21–31.

Hamza, M.S., Reyes, R.A., Izumiya, Y., Wisdom, R., Kung, H.J., Luciw, P.A., 2004.ORF36 protein kinase of Kaposi’s sarcoma herpesvirus activates the c-Jun N-terminal kinase signaling pathway. Journal of Biological Chemistry 279 (37),38325–38330.

Kato, A., Yamamoto, M., Ohno, T., Tanaka, M., Sata, T., Nishiyama, Y., Kawaguchi,Y., 2006. Herpes simplex virus 1-encoded protein kinase UL13 phosphorylatesviral Us3 protein kinase and regulates nuclear localization of viral envelopmentfactors UL34 and UL31. Journal of Virology 80 (3), 1476–1486.

Kramer, A., Liashkovich, I., Oberleithner, H., Ludwig, S., Mazur, I., Shahin, V., 2008.Apoptosis leads to a degradation of vital components of active nuclear transportand a dissociation of the nuclear lamina. Proceedings of the National Academyof Sciences of the United States of America 105 (32), 11236–11241.

Leach, N.R., Roller, R.J., 2010. Significance of host cell kinases in herpes simplexvirus type 1 egress and lamin-associated protein disassembly from the nuclearlamina. Virology 406 (1), 127–137.

Lee, C.P., Huang, Y.H., Lin, S.F., Chang, Y., Chang, Y.H., Takada, K., Chen, M.R., 2008.Epstein–Barr virus BGLF4 kinase induces disassembly of the nuclear lamina tofacilitate virion production. Journal of Virology 82 (23), 11913–11926.

Lotzerich, M., Ruzsics, Z., Koszinowski, U.H., 2006. Functional domains of murinecytomegalovirus nuclear egress protein M53/p38. Journal of Virology 80 (1),73–84.

Manilal, S., Man, N.T., Morris, G.E., 1998. Colocalization of emerin and lamins in inter-phase nuclei and changes during mitosis. Biochemical and Biophysical ResearchCommunications 249, 643–647.

Mendez-Lopez, I., Worman, H.J., 2012. Inner nuclear membrane proteins: impacton human disease. Chromosoma 121 (2), 153–167.

Milbradt, J., Auerochs, S., Sticht, H., Marschall, M., 2009. Cytomegaloviral proteinsthat associate with the nuclear lamina: components of a postulated nuclearegress complex. Journal of General Virology 90 (Pt 3), 579–590.

Morris, J.B., Hofemeister, H., O’Hare, P., 2007. Herpes simplex virus infection inducesphosphorylation and delocalization of emerin, a key inner nuclear membraneprotein. Journal of Virology 81 (9), 4429–4437.

Morrison, L.A., Delassus, G.S., 2011. Breach of the nuclear lamina during assemblyof herpes simplex viruses. Nucleus 2 (4), 271–276.

Mou, F., Wills, E., Baines, J.D., 2009. Phosphorylation of the U(L)31 protein of herpessimplex virus 1 by the U(S)3-encoded kinase regulates localization of the nuclearenvelopment complex and egress of nucleocapsids. Journal of Virology 83 (10),5181–5191.

Nakamura, H., Lu, M., Gwack, Y., Souvlis, J., Zeichner, S.L., Jung, J.U., 2003. Globalchanges in Kaposi’s sarcoma-associated virus gene expression patterns follow-ing expression of a tetracycline-inducible Rta transactivator. Journal of Virology77 (7), 4205–4220.

Pyrpasopoulou, 1996. The lamin B receptor (LBR) provides essential chromatindocking sites at the nuclear envelope. EMBO Journal 24, 11.

Santarelli, R., De Marco, R., Masala, M.V., Angeloni, A., Uccini, S., Pacchiarotti, R.,Montesu, M.A., Satta, R., Cerimele, D., Faggioni, A., Cottoni, F., 2001. Directcorrelation between human herpesvirus-8 seroprevalence and classic Kaposi’ssarcoma incidence in Northern Sardinia. Journal of Medical Virology 65 (2),368–372.

Santarelli, R., Farina, A., Granato, M., Gonnella, R., Raffa, S., Leone, L., Bei, R., Modesti,A., Frati, L., Torrisi, M.R., Faggioni, A., 2008. Identification and characterizationof the product encoded by ORF69 of Kaposi’s sarcoma-associated herpesvirus.Journal of Virology 82 (9), 4562–4572.

Senior, A., Gerace, L., 1988. Integral membrane proteins specific to the inner nuclearmembrane and associated with the nuclear lamina. Journal of Cell Biology 107(6), 2029–2036.

Squarzoni, S., Sabatelli, P., Ognibene, A., Toniolo, D., Cartegni, L., Cobianchi, F., Petrini,S., Merlini, L., Maraldi, N.M., 1998. Immunocytochemical detection of emerinwithin the nuclear matrix. Neuromuscular Disorders: NMD 8 (5), 338–344.

Vaughan, A., Alvarez-Reyes, M., Bridger, J.M., Broers, J.L., Ramaekers, F.C., Wehnert,M., Morris, G.E., Whitfield, W.G.F., Hutchison, C.J., 2001. Both emerin and laminC depend on lamin A for localization at the nuclear envelope. Journal of CellScience 114 (Pt 14), 2577–2590.

Worman, H.J., 2012. Nuclear lamins and laminopathies. Journal of Pathology 226(2), 316–325.

Zhao, J., Punj, V., Matta, H., Mazzacurati, L., Schamus, S., Yang, Y., Yang, T., Hong, Y.,Chaudhary, P.M., 2007. K13 blocks KSHV lytic replication and deregulates vIL6and hIL6 expression: a model of lytic replication induced clonal selection in viraloncogenesis. PloS ONE 2 (10), e1067.