†These authors contributed equally to this publication.Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin a/b and importin b1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartitetype aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K 387 K 388 K 389 ); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)–1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen®) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.

Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.

Although all established functions of dengue virus NS5 (nonstructural protein 5) occur in the cytoplasm, its nuclear localization, mediated by dual nuclear localization sequences, is essential for virus replication. Here, we have determined the mechanism by which NS5 can localize in the cytoplasm to perform its role in replication, establishing for the first time that it is able to be exported from the nucleus by the exportin CRM1 and hence can shuttle between the nucleus and cytoplasm. We define the nuclear export sequence responsible to be residues 327-343 and confirm interaction of NS5 and CRM1 by pulldown assay. Significantly, greater nuclear accumulation of NS5 during infection due to CRM1 inhibition coincided with altered kinetics of virus production and decreased induction of the antiviral chemokine interleukin-8. This is the first report of a nuclear export sequence within NS5 for any member of the Flavivirus genus; because of its high conservation within the genus, it may represent a target for the treatment of diseases caused by several medically important flaviviruses.The four serotypes of dengue virus (DENV-1-4) 2 are the causative agents of the most common arthropod-borne viral disease, dengue fever, and its more severe and potentially deadly dengue hemorrhagic fever form (1). DENV is a member of the genus Flavivirus within the family Flaviviridae. Like all flaviviruses, DENV possesses an ϳ11-kb, positive-sense, singlestranded RNA genome that is translated as one long polyprotein and cleaved into 10 viral proteins: three structural (capsid, pre-membrane/membrane, and envelope) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (2). Flavivirus replication takes place in the cytoplasm, whereby several viral NS and host proteins are believed to constitute the replication complex, the proposed replication machinery of flaviviruses (3). Two key enzymes in replication, NS3 and NS5, the RNA helicase and RNA-dependent RNA polymerase, respectively, interact within the cytoplasm of infected cells (4).The multifunctional NS5 protein is the largest (900 amino acids, 105 kDa) and most highly conserved of the dengue NS proteins (5-7). NS5 contains an N-terminal S-adenosylmethyltransferase domain (5) and a C-terminal RNA-dependent RNA polymerase domain (8 -10) separated by an "interdomain linker region" (see Fig. 1). Despite all well established functions of NS5 occurring within the cytoplasm (2), NS5 is predominantly nuclear in DENV-2-infected cells (4, 11).Proteins Ͼ45 kDa require a nuclear localization sequence (NLS) for transport into the nucleus (12, 13). NLSs confer interaction with members of the importin (Imp) superfamily of transporters (either an Imp-␣/␤ heterodimer or Imp-␤ or a homolog thereof), which mediate the translocation of a cargo into the nucleus. Within the nucleus, the cargo-NLS-Imp complex is dissociated through binding of Ran-GTP to Imp-␤, releasing the cargo into the nucleoplasm. Analogously, proteins containing nuclear export sequences (NESs) interact with Imp-␤ ...

Highlights d Elucidation of the interface of full-length STAT1 and a viral interferon antagonist d The viral protein-STAT1 interface involves multiple distinct surfaces d Ablation of the interaction requires multiple mutations at distinct sites d Loss of viral protein-STAT1 interaction attenuates a rabies virus street strain

Recent studies indicate that nucleoli play critical roles in the DNA-damage response (DDR) via interaction of DDR machinery including NBS1 with nucleolar Treacle protein, a key mediator of ribosomal RNA (rRNA) transcription and processing. Here, using proteomics, confocal and single molecule super-resolution imaging, and infection under biosafety level-4 containment, we show that this nucleolar DDR pathway is targeted by infectious pathogens. We find that the matrix proteins of Hendra virus and Nipah virus, highly pathogenic viruses of the Henipavirus genus in the order Mononegavirales, interact with Treacle and inhibit its function, thereby silencing rRNA biogenesis, consistent with mimicking NBS1–Treacle interaction during a DDR. Furthermore, inhibition of Treacle expression/function enhances henipavirus production. These data identify a mechanism for viral modulation of host cells by appropriating the nucleolar DDR and represent, to our knowledge, the first direct intranucleolar function for proteins of any mononegavirus.

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SummaryIn recent years, understanding of the nucleolus has undergone a renaissance. Once considered primarily as the sites of ribosome biogenesis, nucleoli are now understood to be highly dynamic, multifunctional structures that participate in a plethora of cellular functions including regulation of the cell cycle, signal recognition particle assembly, apoptosis and stress responses. Although the molecular/mechanistic details of many of these functions remain only partially resolved, it is becoming increasingly apparent that nucleoli are also common targets of almost all types of viruses, potentially allowing viruses to manipulate cellular responses and the intracellular environment to facilitate replication and propagation. Importantly, a number of recent studies have moved beyond early descriptive observations to identify key roles for nucleolar interactions in the viral life cycle and pathogenesis. While it is perhaps unsurprising that many viruses that replicate within the nucleus also form interactions with nucleoli, the roles of nucleoli in the biology of cytoplasmic viruses is less intuitive. Nevertheless, a number of positivestranded RNA viruses that replicate exclusively in the cytoplasm are known to express proteins that enter the nucleus and target nucleoli, and recent data have indicated similar processes in several cytoplasmic negative-sense RNA viruses. Here, we review this emerging aspect of the virus-host interface with a focus on examples where virusnucleolus interactions have been linked to specific functional outcomes/mechanistic processes in infection and on the nucleolar interfaces formed by viruses that replicate exclusively in the cytoplasm.