No abstract

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway.The influenza virus genome consists of eight segments of single-stranded RNA that encode a total of 10 identified polypeptides. The genomic RNA segments are of negative sense and are always found in association with viral polypeptides: the three subunits of an RNA-dependent RNA polymerase (PB1, PB2, and PA) and, in stoichiometric quantities, a single-strand RNA-binding nucleoprotein (NP) (28). In virions, these ribonucleoprotein (RNP) structures are packaged within a shell of the viral M1 polypeptide underlying the lipid bilayer, along with the hemagglutinin (HA) and neuraminidase integral membrane glycoproteins. Minor virion components include M2, a small transmembrane ion channel, and the NS2 polypeptide (28). Influenza virus particles enter the cell by receptor-mediated endocytosis. Following acidification of the endosome, the M1 polypeptide dissociates from the RNP segments and virion RNPs (vRNPs) are released into the cytoplasm (30, 31). Unusually for a virus with no DNA coding stage, influenza virus transcription occurs in the nucleus (20,22). Accordingly, after release of the RNPs into the cytoplasm, they migrate into the nucleus, in an active process that is thought to be mediated by the cellular importin ␣/␤ pathway (39). Once in the nucleus, vRNPs act as the template for synthesis of mRNAs, which are exported into the cytoplasm for translation. The vRNPs also act as the template for synthesis of full-length cRNA co...

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.Herpes simplex virus 1 (HSV-1) forms replication compartments (RCs) in the infected cell nucleus (32), in which DNA replication, late viral transcription, and viral nucleocapsid assembly occur. In doing so, the virus causes cytopathic effects by affecting factors that control nuclear architecture: host cell chromatin and the nuclear lamina (5,24,34,40,41). During infection, RCs form from small prereplicative sites and expand into large globular domains, disrupting the nuclear interior by compressing and marginalizing host chromatin (24,39,44,45). Following assembly, nucleocapsids are thought to exit the nucleus by budding at the inner nuclear membrane into the perinuclear space (11). This requires that nucleocapsids move through the host chromatin layer and the nuclear lamina to reach the membrane. Thus, HSV-1 manipulates the nuclear interior and periphery to achieve replication and egress.Several studies have described changes in the nuclear lamina during infection with different herpesviruses (9,25,34,40,41). Mouse cytomegalovirus has been shown to disrupt the nuclear lamina, and two viral proteins, UL50 and UL53, have been implicated in this process (25). Homologues of these proteins appear to be present in several other viruses, including HSV-1, HSV-2, pseudorabies virus, and Epstein-Barr virus (EBV) (19,21,25,35,51). In EBV, the proteins BFLF2 and BFRF1 have been shown to interact and colocalize at the nuclear membrane, and BFRF1 binds to lamin B in vitro (9, 21). A mutant EBV lacking a functional BFRF1 gene is defective for replication in several cell lines and shows accumulation of nucleocapsids in the nucleus of infected cells (6). The homologous pseudorabies virus UL31 and UL34 proteins have been shown to interact with one ano...

Morphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: approximately 100-nm spheres and similar diameter filaments that reach 20 microm in length. Spherical particles assemble at plasma membrane lipid rafts in a process independent of microfilaments. The budding site of filamentous virions is hitherto uncharacterised but their formation involves the actin cytoskeleton. We confirm microfilament involvement in filamentous budding and show that after disruption of cortical actin by jasplakinolide, HA, NP, and M1 redistributed around beta-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4 degrees C. Furthermore, in both cases HA partitioned into low buoyant density detergent-insoluble glycolipid domains, indicating that filamentous virions and annuli contain reorganised lipid rafts. We propose that the actin cytoskeleton is required to maintain the correct organisation of lipid rafts for incorporation into budding viral filaments.