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Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCFSkp2 ubiquitin ligase. In these studies, we explored its roles in estrogen receptor–positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G1 by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCFSkp2 target. Heregulin β1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCFSkp2 target, p27Kip1. Remarkably, however, Cks1-depleted cells not only exhibit slowed G1 progression, but also accumulate in G2-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G2-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin β1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer. [Cancer Res 2007;67(23):11393–401]

Supplementary Figure 2 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Figure 4 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Table 1 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Figure 1 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Figure Legends 1-4 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Supplementary Figure 3 from Cks1 Regulates cdk1 Expression: A Novel Role during Mitotic Entry in Breast Cancer Cells

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97., While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.

Luspatercept is a recombinant fusion protein that binds and sequesters several endogenous transforming growth factor-beta superfamily ligands, including growth differentiation factor 11, thereby diminishing Smad2/3 signaling in target cells involved in erythropoiesis. Luspatercept, and its murine analog RAP-536, have been shown to act as erythroid maturation agents via their effects on late-stage erythropoiesis by inducing erythroblast maturation, leading to increases in red blood cells (RBCs) and hemoglobin (Hb). This study demonstrated that thalassemic (th3/+) reticulocytes are unstable, and that RAP-536, in addition to its function as an erythroid maturation agent, modulates the maturation of wild-type (WT) and th3/+ reticulocytes. Furthermore, RAP-536 treatment increased RBCs and decreased bilirubin in a mouse model of alpha-thalassemia (129S-Hba-a1tm1Led/J). To examine whether acute RAP-536 treatment acts on reticulocytes and alters reticulocyte levels in blood, the blood of WT mice was analyzed 3, 12, and 24 hours, and 2, 3, 4, and 7 days after a single dose of RAP-536 (10 or 30 mg/kg) or vehicle. RAP-536 treatment increased RBCs, Hb, and hematocrit significantly at all time points, compared with vehicle. However, in mice treated with RAP-536, reticulocytes in blood decreased significantly on Days 2, 3, and 4 and returned to normal baseline levels on Day 7. Analysis of reticulocyte subpopulations in blood 3 days after RAP-536 treatment showed that the relative percentages of immature reticulocytes (CD71+ or high RNA content) within the blood reticulocyte population decreased, suggesting that reticulocytes released from the bone marrow (BM) were more mature and/or reticulocytes matured faster in blood. A quantitative pharmacology (QP) model was developed to explore which RAP-536-induced modulations of erythropoiesis in WT mice can simulate the experimental observations. The model represents erythroblast, reticulocyte, and RBC (erythrocyte) maturation stages in BM, peripheral blood, and spleen, in the presence or absence of a RAP-536 effect. The QP model consists of a system of ordinary differential equations, with homeostatic parameter values assigned from literature or experimental measures, and RAP-536-perturbed parameter values regressed by fitting the model to erythropoiesis data of RAP-536-treated WT mice. Comparison of model parameters for homeostatic versus RAP-536-perturbed states indicated that RAP-536 leads to an increase in the erythroblast-to-reticulocyte and reticulocyte-to-RBC conversion rates, the transfer of BM reticulocytes to blood, and a delayed increase in erythroblast production. To directly test whether RAP-536 treatment affects reticulocyte development in blood, comparative blood transfusion experiments were performed. Biotinylated GFP+ blood from WT mice (C57BL/6-Tg(UBC-GFP)30Scha/J) and biotinylated GFP− blood from th3/+ beta-thalassemic mice (B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J) were co-transfused into GFP− WT recipient mice (C57BL/6J), which were subsequently treated with RAP-536 or vehicle. In the donor reticulocyte population, th3/+ reticulocyte percentage decreased continuously up to 3 days after transfusion, suggesting that many of the th3/+ reticulocytes were eliminated before they could form RBCs. However, compared with vehicle, RAP-536 treatment led to increased persistence of the relative percentages of th3/+ reticulocytes (Figure A). Consequently, 7 days after transfusion, when most reticulocytes have matured to RBCs, the percentage of th3/+ RBC among donor RBCs was higher with RAP-536 (Figure B). Finally, treatment of an alpha-thalassemia mouse model (129S-Hba-a1tm1Led/J) with RAP-536 10 mg/kg for 8 weeks increased RBCs and hematocrit and reduced serum bilirubin, compared with vehicle. These results suggest that RAP-536 is, as previously shown, an erythroid maturation agent, which also modulates reticulocyte maturation in blood. In WT mice, RAP-536 modulated blood reticulocyte dynamics consistent with faster maturation. RAP-536 also prolonged the persistence of th3/+ reticulocytes and maintained a higher frequency of th3/+ RBCs. These data, together with the finding that RAP-536 reduces hemolysis in an experimental alpha-thalassemia disease model, suggest that luspatercept has the potential to improve anemias associated with hemolysis and/or reticulocytosis. Disclosures Acar: Bristol Myers Squibb: Ended employment in the past 24 months. Jupelli:Bristol Myers Squibb: Current Employment. Abbiati:Bristol Myers Squibb: Current Employment. Ramanathan:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Santini:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ratushny:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Dunshee:Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Genentech Inc.: Current Employment, Current equity holder in publicly-traded company. Lopes de Menezes:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. MacBeth:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Suragani:Acceleron Pharma: Current Employment, Current equity holder in publicly-traded company. Loos:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Schwickart:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the post-fusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E expression; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. YFVΔSK/Nluc gene expression was also shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur, as well as valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a post-fusion, pre-translation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a post-fusion step in virus entry.IMPORTANCEFlaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here we show that the yellow fever virus nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

Post-translational modification of proteins with ubiquitin regulates a variety of eukaryotic cellular processes. Ubiquitin can be conjugated to substrates either as a single moiety (monoubiquitination) or as isopeptide bond-linked chains (polyubiquitination), creating an array of ubiquitin signals. It has been established that monoubiquitination can serve important functions in many biological processes such as the regulation of gene transcription, protein trafficking, and DNA repair. Surprisingly, little is known about the mechanisms by which monoubiquitin signals are produced in the cell. Here, we discuss the potential cellular strategies for generating monoubiquitinated proteins using a few, relatively well characterized examples of monoubiquitinated proteins. These strategies include coupling ubiquitination to low affinity ubiquitin binding, using monoubiquitination-dedicated E2 conjugating enzymes, and restricting ubiquitin chain elongation. Some of these principles may be applicable to protein modifications involving ubiquitin like proteins (UBLs), which often occur in monomeric form.

To replicate its segmented, double-stranded RNA (dsRNA) genome, the rotavirus RNA-dependent RNA polymerase, VP1, must recognize viral plus-strand RNAs (+RNAs) and guide them into the catalytic center. VP1 binds to the conserved 3′ end of rotavirus +RNAs via both sequence-dependent and sequence-independent contacts. Sequence-dependent contacts permit recognition of viral +RNAs and specify an autoinhibited positioning of the template within the catalytic site. However, the contributions to dsRNA synthesis of sequence-dependent and sequence-independent VP1-RNA interactions remain unclear. To analyze the importance of VP1 residues that interact with +RNA on genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro . Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutations led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of +RNA templates. In contrast, point mutations of sequence-independent RNA contact residues led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with +RNA to mediate template recognition and dsRNA synthesis yet function in concert to promote viral RNA replication at appropriate times and rates.

Recently we reported that the N protein of the Old World hantavirus, Hantaan (HTNV), traffics on microtubules to the ER-Golgi intermediate compartment (ERGIC) prior to its movement to the Golgi and requires an intact ERGIC for viral replication. We have extended these studies to the New World hantaviruses, Andes virus (ANDV) and Black Creek Canal virus (BCCV), and an additional Old World hantavirus, Seoul virus (SEOV). These studies support microtubule-dependent trafficking of the N protein to ERGIC within the perinuclear region for the New and Old World hantaviruses. However, we observed that early entry events were distinct for HTNV and ANDV with respect to the pathway for entry and the dependence on an intact actin (ANDV) versus microtubule (HTNV) cytoskeleton for viral replication. These studies show for the first time that while Old and New World hantaviruses share common features in their pathways, they have evolved differences in their interaction with host cell machinery.

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly.

In contrast to most negative-stranded RNA viruses, hantaviruses and other viruses in the family Bunyaviridae mature intracellularly, deriving the virion envelope from the endoplasmic reticulum (ER) or Golgi compartment. While it is generally accepted that Old World hantaviruses assemble and bud into the Golgi compartment, some studies with New World hantaviruses have raised the possibility of maturation at the plasma membrane as well. Overall, the steps leading to virion assembly remain largely undetermined for hantaviruses. Because hantaviruses do not have matrix proteins, the nucleocapsid protein (N) has been proposed to play a key role in assembly. Herein, we examine the intracellular trafficking and morphogenesis of the prototype Old World hantavirus, Hantaan virus (HTNV). Using confocal microscopy, we show that N colocalized with the ER-Golgi intermediate compartment (ERGIC) in HTNV-infected Vero E6 cells, not with the ER, Golgi compartment, or early endosomes. Brefeldin A, which effectively disperses the ER, the ERGIC, and Golgi membranes, redistributed N with the ERGIC, implicating membrane association; however, subcellular fractionation experiments showed the majority of N in particulate fractions. Confocal microscopy revealed that N was juxtaposed to and distributed along microtubules and, over time, became surrounded by vimentin cages. To probe cytoskeletal association further, we probed trafficking of N in cells treated with nocodazole and cytochalasin D, which depolymerize microtubules and actin, respectively. We show that nocodazole, but not cytochalasin D, affected the distribution of N and reduced levels of intracellular viral RNA. These results suggested the involvement of microtubules in trafficking of N, whose movement could occur via molecular motors such as dynein. Overexpression of dynamitin, which is associated with dynein-mediated transport, creates a dominant-negative phenotype blocking transport on microtubules. Overexpression of dynamitin reduced N accumulation in the perinuclear region, which further supports microtubule components in N trafficking. The combined results of these experiments support targeting of N to the ERGIC prior to its movement to the Golgi compartment and the requirement of an intact ERGIC for viral replication and, thus, the possibility of virus factories in this region.

The rotavirus RNA-dependent RNA polymerase (RdRp), VP1, contains canonical RdRp motifs and a priming loop that is hypothesized to undergo conformational rearrangements during RNA synthesis. In the absence of viral core shell protein VP2, VP1 fails to interact stably with divalent cations or nucleotides and has a retracted priming loop. To identify residues of potential import to nucleotide and divalent cation stabilization, we aligned VP1 of divergent rotaviruses and the structural homolog reovirus λ3. VP1 mutants were engineered and characterized for RNA synthetic capacity in vitro. Conserved aspartic acids in RdRp motifs A and C and arginines in motif F that likely stabilize divalent cations and nucleotides were required for efficient RNA synthesis. Mutation of individual priming loop residues diminished or enhanced RNA synthesis efficiency without obviating the need for VP2, which suggests that this structure serves as a dynamic regulatory element that links RdRp activity to particle assembly.

Cks1 plays an essential role in SCFSkp2-mediated ubiquitination, and consequently turnover, of the cdk2 inhibitor and tumor supressor p27Kip1. High Cks1 expression is associated with aggressive breast tumors and correlates with low p27Kip1 levels in some cases, although it is also an independent prognostic marker for survival, and provides predictive information in addition to that provided by p27Kip1 alone. In this report we demonstrate that Cks1 protein and mRNA are elevated to very high levels in mammary tumors initiated by erbB2, c-myc and polyoma middle-T (PyMT) in transgenic mice, whereas Cks1 protein is hardly detectable in the normal mammary epithelium. Cks1 is also highly upregulated in rat mammary tumors initiated by methylnitrosourea (MNU). Despite high levels of Cks1 expression, p27Kip1 levels were not reduced, and were in fact slightly higher in mammary tumors initiated by erbB2, PyMT and MNU. In contrast mammary tumors from MMTV-c-myc mice did exhibit low p27Kip1 and higher levels of Skp2. Together, these data suggest that deregulated Cks1 expression might play roles in oncogene and carcinogen-initiated mammary tumorigenesis independent of p27Kip1 turnover in certain tumors. Stable overexpression of Cks1 in human breast carcinoma MCF-7 cells did not significantly reduce p27Kip1 expression, although it conferred resistance to Faslodex (ICI 182780)-mediated inhibition of colony outgrowth in these cells. In contrast, Cks1-depleted MCF-7 cells formed fewer colonies in estrogen-containing medium. Therefore, our studies also suggest that Cks1 levels regulate the responsiveness of ER+ breast cancers to estrogens and anti-estrogens.

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

Cks1, a small protein whose expression is strongly associated with aggressive breast tumors, is a component of cyclin-cdk complexes, as well as the SCFSkp2 ubiquitin ligase. In these studies, we explored its roles in estrogen receptor–positive breast tumor cells. When exposed to the antiestrogen ICI 182780, these cells accumulate in G1 by reducing the expression of Cks1, and increasing the levels of p130/Rb2, a cdk2 inhibitor and SCFSkp2 target. Heregulin β1 or estradiol abrogate antiestrogen effects by increasing Cks1 expression, down-regulating p130/Rb2 and inducing S phase entry. Depletion of Cks1 in these cells by RNA interference concomitantly decreased Skp2 and up-regulated p130/Rb2 and another SCFSkp2 target, p27Kip1. Remarkably, however, Cks1-depleted cells not only exhibit slowed G1 progression, but also accumulate in G2-M due to blocked mitotic entry. Notably, we show that cdk1 expression, which is crucial for M phase entry, is drastically diminished by Cks1 depletion, and that restoration of cdk1 reduces G2-M accumulation in Cks1-depleted cells. cdk1 reduction in Cks1-depleted cells is a consequence of a marked decrease in its mRNA and not due to alteration in its proteolytic turnover. Both heregulin β1 and estradiol could neither restore cdk1 nor sustain cycling in Cks1-depleted cells, although classical estrogen receptor function remained unaltered. Cks1 depletion also decreased Skp2 in human mammary epithelial cells without altering cell cycle progression. Thus, the indispensability of Cks1 to the breast cancer cell cycle, versus its redundancy in normal cells, suggests that Cks1 abrogation could be an effective interventional strategy in breast cancer. [Cancer Res 2007;67(23):11393–401]

Hepatitis C virus (HCV) infection is a leading cause of liver disease worldwide. The HCV RNA genome is translated into a single polyprotein. Most of the cleavage sites in the non-structural (NS) polyprotein region are processed by the NS3/NS4A serine protease. The vital NS2-NS3 cleavage is catalyzed by the NS2 autoprotease. For efficient processing at the NS2/NS3 site, the NS2 cysteine protease depends on the NS3 serine protease domain. Despite its importance for the viral life cycle, the molecular details of the NS2 autoprotease activation by NS3 are poorly understood. Here, we report the identification of a conserved hydrophobic NS3 surface patch that is essential for NS2 protease activation. One residue within this surface region is also critical for RNA replication and NS5A hyperphosphorylation, two processes known to depend on functional replicase assembly. This dual function of the NS3 surface patch prompted us to reinvestigate the impact of the NS2-NS3 cleavage on NS5A hyperphosphorylation. Interestingly, NS2-NS3 cleavage turned out to be a prerequisite for NS5A hyperphosphorylation, indicating that this cleavage has to occur prior to replicase assembly. Based on our data, we propose a sequential cascade of molecular events: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. This model explains why efficient NS2-3 cleavage is pivotal for HCV RNA replication. According to our model, the hydrophobic surface patch on NS3 represents a module critically involved in the temporal coordination of HCV replicase assembly., Author Summary Hepatitis C virus (HCV) replicates its genome in close association to cellular membranes which serve as assembly site of multi-subunit replication complexes. The process of replication complex maturation must be properly controlled to prevent the non-functional maturation/assembly of these complexes. In this process, the temporal regulation of viral polyprotein processing often plays a pivotal role as exemplified by the strict requirement for NS2-NS3 cleavage for HCV genome replication. We demonstrate here that a conserved hydrophobic NS3 surface patch activates the NS2 protease to stimulate NS2-NS3 cleavage. By dissecting the role of these NS3 surface residues in viral RNA replication, we show that one of these NS3 residues is also a critical determinant for HCV genome replication by negatively regulating NS5A hyperphosphorylation. Surprisingly, further experiments revealed that the NS2-NS3 cleavage is a prerequisite for NS5A hyperphosphorylation. To fulfill the requirements for gradual assembly into functional replication complexes, an ordered cascade of molecular events takes place: in uncleaved NS2-NS3, the hydrophobic NS3 surface patch promotes NS2 protease stimulation; upon NS2-NS3 cleavage, this surface region becomes available for functional replicase assembly. As a consequence, the hydrophobic surface patch on free NS3 can promote NS5A hyperphosphorylation as an indication of functional replicase assembly.

Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors including CDC-45 and GINS. During the elongation step the CDC-45/GINS complex moves with the replication fork, however it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48UFD-1/NPL-4 deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.

Background Early endosomal autoantigen 1 (EEA1) is a membrane tethering factor required for the fusion and maturation of early endosomes in endocytosis. How the activity of EEA1 is regulated in cells is unclear. Results Here we show that endogenous EEA1 is prone to monoubiquitination at multiple sites, owing to an intrinsic affinity to ubiquitin conjugating enzymes (E2). The E2 interactions enable a ubiquitin ligase (E3) independent mechanism that decorate EEA1 with multiple mono-ubiquitin moieties. Expression of an ubiquitin-EEA1 chimera that mimics native mono-ubiquitinated EEA1 generates giant endosomes abutting the nucleus. Several lines of evidence suggest that this phenotype is due to increased endosome fusion and a simultaneous blockade on an endosome recycling pathway. The latter is likely caused by diminished endosome fission in cells expressing ubiquitin-EEA1. Conclusion Our results demonstrate that ubiquitination may dramatically affect the activity of an endosome fusion factor to alter endosome morphology and trafficking pattern, and thereby implicating an unexpected role of ubiquitin signaling in endocytosis.