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

Abstract

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...

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

“…The ring-like structure is also of interest when considering further collective functions of the NEC. In general, the HCMV-specific NEC not only clusters proteins to distinct membrane sites and induces the formation of locally restricted lamina-depleted areas (13), but also offers multiple attachment points for additional quaternary protein-protein interactions. Thus, the highly ordered ring-like arrangement might promote the controlled association of further NEC proteins.…”

Modification of the major tegument protein pp65 of human cytomegalovirus inhibits virus growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells

“…replication (Wolf et al, 2001) and nuclear capsid egress (Hamirally et al, 2009;Krosky et al, 2003;Marschall et al, 2005;Milbradt et al, 2010). Although DNA replication was unimpaired in RV-VM1-infected cells, a possible explanation of the phenotypic alterations was that pp65-VM1 interfered with essential activities of pUL97 at later stages.…”

The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import

“…pUL50, pUL53, p32, lamin B receptor (LBR), PKC and pUL97 (Marschall et al, 2005(Marschall et al, , 2011Muranyi et al, 2002). Other components of the nuclear envelope, such as lamins of type A, as well as regulatory factors, such as the prolyl isomerase Pin1, were also postulated to belong to the group of NEC-associated proteins (Milbradt et al, 2010).…”