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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.

Herpes simplex virus 1 (HSV-1) forms replication compartments (RCs), domains in which viral DNA replication, late-gene transcription, and encapsidation take place, in the host cell nucleus. The formation of these domains leads to compression and marginalization of host cell chromatin, which forms a dense layer surrounding the viral RCs and constitutes a potential barrier to viral nuclear egress or primary envelopment at the inner nuclear membrane. Surrounding the chromatin layer is the nuclear lamina, a further host cell barrier to egress. In this study, we describe an additional phase in RC maturation that involves disruption of the host chromatin and nuclear lamina so that the RC can approach the nuclear envelope. During this phase, the structure of the chromatin layer is altered so that it no longer forms a continuous layer around the RCs but instead is fragmented, forming islands between which RCs extend to reach the nuclear periphery. Coincident with these changes, the nuclear lamina components lamin A/C and lamin-associated protein 2 appear to be redistributed via a mechanism involving the U L 31 and U L 34 gene products. Viruses in which the U L 31 or U L 34 gene has been deleted are unable to undergo this phase of chromatin reorganization and lamina alterations and instead form RCs which are bounded by an intact host cell chromatin layer and nuclear lamina. We postulate that these defects in chromatin restructuring and lamina reorganization explain the previously documented growth defects of these mutant viruses.Herpes simplex virus 1 (HSV-1) is a large double-stranded DNA virus which replicates in the nucleus of infected host cells. Viral DNA replication and late gene transcription occur in replication compartments (RCs), nuclear domains which are formed when viral replication successively annexes large portions of the nucleus during the early stages of infection. During the later stages of replication, assembly of viral capsids occurs and DNA packaging takes place in RCs. Following this, capsids move to the inner nuclear membrane (INM), where primary envelopment takes place as they bud into the perinuclear space, acquiring an envelope derived from the INM. Herpesvirus particles are then thought to fuse with the outer nuclear membrane, releasing viral nucleocapsids into the host cell cytosol (17, 28). Finally, the nucleocapsid undergoes secondary envelopment and egress from the cell (24).During RC formation and annexation of space in the host cell nucleus, cellular chromatin is marginalized and compressed (18,26). This results in a layer of host cell chromatin surrounding the RC, which potentially constitutes a barrier through which viral capsids must move to reach the INM. Thus, to bud through the INM, viral nucleocapsids must move through the compacted host chromatin and the nuclear lamina. The nuclear lamina, a proteinaceous layer underlying the INM, is composed of integral INM proteins such as lamin-associated protein 2 (LAP2) and lamin B receptor, which interact with lamin proteins A, B, and C (2, ...

Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (⌬E3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2␣ phosphorylation and impaired translation initiation. Here we show that cells infected with ⌬E3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ⌬E3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2␣, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2␣ showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ⌬E3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ⌬E3L mutant VV, despite increased eIF2␣ phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ⌬E3L mutant VV replication requires AVG formation subsequent to eIF2␣ phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.Eukaryotic cells have evolved stress-responsive pathways to cope with various environmental challenges. A central feature of the cellular stress response is the reprogramming of mRNA translation (21). One of several signaling pathways that control translation during stress is the eIF2␣ pathway, which regulates the recruitment of the initiator methionine by the translation initiation factor eIF2. A family of protein kinases phosphorylate a common site in eIF2␣ (serine 51), the regulatory subunit of eIF2. This phosphorylation inhibits eIF2 function, limiting preinitiation complex formation and reducing translation initiation (48). Each of the eIF2␣ kinases, which include protein kinase R (PKR), heme-regulated eIF2␣ kinase, general control nonderepressible 2, and PKR-like endoplasmic reticulum (ER) kinase, is activated in response to different environmental stresses such as oxidative and ER stress, heat shock, amino acid deprivation, and hemin deficiency. Some are also activated by immune signaling cascades and/or assault by pathogens, including viruses (26,31,37). A key consequence of eIF2␣ phosphorylation is the formation of cytoplasmic stress granules (SGs) (4), foci in which stalled mRNP (messenger ribonucleoprotein) complexes accumulate f...

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperaturesensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.The influenza A virus nucleoprotein (NP) is a 56-kDa basic RNA-binding protein encoded by segment 5 that plays an essential structural role, encapsidating the segmented viral genome into ribonucleoproteins (RNPs). RNPs are helical structures consisting of the viral-RNA (vRNA)-dependent RNA polymerase and a chain of NP monomers around which the negative-sense single-stranded vRNA segments are wrapped (56). In the early stages of the replication cycle, infecting RNPs are imported into the nucleus, where they are transcribed and replicated. There is much evidence that NP has essential functions during this period of the viral life cycle. Nuclear localization signals in the protein are sufficient to direct nuclear import of the genome (53). Once in the nucleus, NP is essential for vRNA synthesis (30). NP encapsidates the genome through a sequence-independent RNA-binding activity (58, 67) and interactions with the viral polymerase (8,48,55). Coating of the genomic vRNA segments by NP is probably necessary for synthesis of long RNA products by the viral polymerase (29), although it is not required for the synthesis of short products (37). NP has also long been associated with a specific requirement for genome replication (7, 61), although recent research suggests this may be more as a facilitator than as a regulator (48,50,65).During the later stages of infection, RNPs traffic through ...