Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex

Abstract

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...

Novel Mode of Phosphorylation-triggered Reorganization of the Nuclear Lamina during Nuclear Egress of Human Cytomegalovirus

“…pcDNA-UL97-FLAG, pcDNA-UL97(K355M)-FLAG, pcDNA-UL97-(1-595)-FLAG, and pcDNA-UL97-(181-707)-FLAG)), hemagglutinin-tagged pUL50 (pcDNA-UL50-HA), and PKC␣ fused to enhanced green fluorescent protein (EGFP; pEGFP-N1-PKC␣) were described previously (19,21,22,27). Plasmid pHM990, coding for a fusion protein of IE2p86 and GFP, was kindly provided by Dr. N. Tavalai (Institute for Clinical and Molecular Virology, University of Erlangen-Nuremberg) (28).…”

“…In particular, the direct and indirect recruitment of PKC and pUL97, respectively, is mediated by viral pUL50. Whereas pUL50 is principally sufficient for PKC recruitment (21), other viral proteins such as pUL53 appear to enhance this function. pUL50 and pUL53 represent an interactor pair of essential nuclear egress proteins that are conserved among herpesviruses (5,18,19,25,26).…”

“…In HCMVinfected cells, the lamina-reorganizing activity of PKC appears to be supported by further lamin-phosphorylating protein kinases. The HCMV-encoded protein kinase pUL97 is recruited to the nuclear lamina (21,22) and is highly important for efficient export of viral capsids to the cytoplasm (23,24). In particular, pUL97 phosphorylates lamins and has a destabilizing effect on the integrity of the nuclear lamina (22).…”

“…This was causatively linked with morphological alterations of the nuclear lamina. On the basis of these observations, we postulated the formation of an HCMV-specific nuclear egress complex (NEC) composed of viral and cellular components (21). This complex may basically consist of pUL50, pUL53, pUL97, PKC, p32, and the lamin B receptor (21).…”

“…On the basis of these observations, we postulated the formation of an HCMV-specific nuclear egress complex (NEC) composed of viral and cellular components (21). This complex may basically consist of pUL50, pUL53, pUL97, PKC, p32, and the lamin B receptor (21). In particular, the direct and indirect recruitment of PKC and pUL97, respectively, is mediated by viral pUL50.…”