Human cytomegalovirus-encoded pUL50 and pUL53 belong to a group of conserved herpesviral nuclear proteins. This study describes: (i) the co-localization of pUL50 with components of the nuclear lamina such as lamins A/C and lamin B receptor by double immunofluorescent staining, (ii) a strong pUL50-mediated relocalization of pUL53 from a diffuse nuclear pattern towards a nuclear rim localization, (iii) a direct interaction between pUL50 and pUL53, as well as between pUL50 and protein kinase C (PKC), shown by yeast two-hybrid and co-immunoprecipitation analyses, (iv) in vitro phosphorylation of pUL50, which is highly suggestive of PKC activity, and finally (v) partial relocalization of PKC by pUL50/pUL53 from its main cytoplasmic localization to a marked nuclear lamina accumulation. These data suggest a role for pUL50 and pUL53 in the recruitment of PKC, an event that is considered to be important for cytomegalovirus-induced distortion of the nuclear lamina. INTRODUCTIONHuman cytomegalovirus (HCMV; family Herpesviridae, subfamily Betaherpesvirinae, genus Cytomegalovirus, species Human herpesvirus 5) is a clinically important, ubiquitous human pathogen, causing severe systemic disease in immunosuppressed patients and prenatally infected children. The most frequently applied antiviral therapies are based on treatment with nucleoside analogues such as ganciclovir (GCV), which is activated by the GCVphosphorylating potential of the UL97-encoded viral kinase (pUL97) (Curran & Noble, 2001).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). ...

The nucleocytoplasmic egress of viral capsids is a rate-limiting step in the replication of the human cytomegalovirus (HCMV). As reported recently, an HCMV-specific nuclear egress complex is composed of viral and cellular proteins, in particular protein kinases with the capacity to induce destabilization of the nuclear lamina. Viral protein kinase pUL97 and cellular protein kinase C (PKC) play important roles by phosphorylating several types of nuclear lamins. Using pUL97 mutants, we show that the lamin-phosphorylating activity of pUL97 is associated with a reorganization of nuclear lamin A/C. Either pUL97 or PKC has the potential to induce distinct punctate lamina-depleted areas at the periphery of the nuclear envelope, which were detectable in transiently transfected and HCMV-infected cells. Using recombinant HCMV, which produces green fluorescent protein-labeled viral capsids, the direct transition of viral capsids through these areas could be visualized. This process was sensitive to an inhibitor of pUL97/PKC activity. The pUL97-mediated phosphorylation of lamin A/C at Ser 22 generated a novel binding motif for the peptidyl-prolyl cis/trans-isomerase Pin1. In HCMV-infected fibroblasts, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to viral replication centers and to the nuclear lamina. The local increase in Pin1 peptidyl-prolyl cis/trans-isomerase activity may promote conformational modulation of lamins. Thus, we postulate a novel phosphorylation-triggered mechanism for the reorganization of the nuclear lamina in HCMVinfected cells. Human cytomegalovirus (HCMV)2 belongs to the ␤-herpesvirus subfamily, exhibiting worldwide distribution. When infecting immunocompetent individuals, HCMV possesses low pathogenicity, causing mainly asymptomatic infections. In immunocompromised or immunosuppressed hosts, HCMV infection can cause severe and even life-threatening diseases, including pneumonitis, retinitis, hepatitis, encephalitis, and gastroenteritis (1-3).HCMV replication is based on a nuclear phase, a characteristic of most DNA viruses. Transition from the nuclear to the cytoplasmic phase is determined by the nuclear exit of DNAfilled capsids budding through the inner nuclear membrane (INM) (4 -6). The site-specific budding of viral capsids through distinct locally occurring invaginations in the INM of HCMVinfected cells was clearly illustrated by Buser et al. (7) using electron microscopic analysis. The proteinaceous network of the nuclear lamina, underlying the INM, constitutes a major obstacle for the nuclear egress of capsids. Lamins, belonging to type V intermediate filament proteins, are the main constituents of the nuclear lamina and are grouped into A and B types. A-type lamins (A, C, A⌬10, and C2; collectively lamin A/C) result from alternative splicing of the LMNA gene. B-type lamins are encoded by the LMNB1 (B1) or LMNB2 (B2, B3) gene (8, 9). A major function of the nuclear lamina is to maintain the structure of the nuclear envelope. During mitosis, the nuclear lamin...

The nuclear egress of cytomegaloviral capsids traversing the nuclear envelope is dependent on a locally restricted destabilization of the rigid nuclear lamina. It has been suggested that the multicomponent nuclear egress complex (NEC) that is formed is comprised of both viral and cellular proteins which act to recruit lamin-phosphorylating protein kinases. Recently, we reported that the lamina-associated human cytomegalovirus-encoded proteins pUL50 and pUL53, conserved among herpesviruses, interact with each other and recruit protein kinase C (PKC) to the nuclear envelope in transfected cells. The multiple interactions of the transmembrane protein pUL50 with pUL53, PKC and cellular PKC-binding protein p32, appear crucial to the formation of the NEC. In this study, we mapped individual interaction sequence elements of pUL50 by coimmunoprecipitation analysis of deletion mutants and yeast two-hybrid studies. Amino acids 1-250 were shown to be responsible for interaction with pUL53, 100-280 for PKC and 100-358 for p32. Interestingly, p32 specifically interacted with multiple NEC components, including the kinases PKC and pUL97, thus possibly acting as an adaptor for protein recruitment to the lamin B receptor. Notably, p32 was the only protein that interacted with the lamin B receptor. Immunofluorescence studies visualized the colocalization of NEC components at the nuclear rim in coexpression studies. The data imply that a tight interaction between at least six viral and cellular proteins leads to the formation of a postulated multi-protein complex required for nuclear egress. INTRODUCTIONHuman cytomegalovirus (HCMV), a member of the bherpesvirus subfamily, is a ubiquitous, clinically important human pathogen that causes severe systemic disease in immunosuppressed hosts and prenatally infected children (Mocarski et al., 2007). As is characteristic for most DNA viruses, HCMV replicates its genome in the nucleus of the host cell. Thereafter, preformed viral capsids are packaged with genomic DNA and have to be transported to the cytoplasm for final maturation and viral release. Due to the large size of cytomegaloviral capsids (~130 nm; Chen et al., 1999), these cannot be transported through the nuclear pore complex (~40 nm; Panté & Kann, 2002). The most widely accepted model for nuclear egress of HCMV and other herpesviruses is based on a transient primary envelopment by budding through the inner nuclear membrane (Mettenleiter, 2004(Mettenleiter, , 2006Sanchez & Spector, 2002;Sampaio et al., 2005). However, before herpesvirusencoded capsids gain access to the inner nuclear membrane, the rigid proteinaceous network of the nuclear lamina provides a major obstacle. Thus, the locally restricted destabilization of the nuclear lamina is required during the viral replication process .A basic principle of the reorganization of the nuclear lamina is the recruitment of cellular and/or virus-encoded protein kinases to increase the site-specific phosphorylation of nuclear lamins and lamin-binding proteins (Dechat et al., 2008). Ce...

Background:The conserved cytomegalovirus proteins pUL50 and pUL53 heterodimerize and form a core nuclear egress complex. Results: The crystal structure of pUL50-pUL53 was solved at 2.44 Å resolution, revealing an N-terminal hooklike extension of pUL53. Conclusion: Data unravel the core NEC architecture, providing a scaffold for viral-cellular NEC protein interactions. Significance: The identified NEC structure will stimulate the development of novel antiviral strategies.