The envelope glycoprotein domain III of dengue virus serotypes 1 and 2 inhibit virus entry

Dengue virus (DV) is a flavivirus and its urban transmission is maintained largely by its mosquito vectors and vertebrate host, often human. In this study, investigation was carried out on the involvement of domain III of the envelope (E) glycosylated protein of dengue virus serotypes 1 and 2 (DV-1 and DV-2 DIII) in binding to host cell surfaces, thus mediating virus entry. Domain III protein of flavivirus can also serve as an attractive target in inhibiting virus entry. The respective DV DIII proteins were expressed as soluble recombinant fusion proteins before purification through enzymatic cleavage and affinity purification. The purified recombinant DV-1 and DV-2 DIII proteins both demonstrated the ability to inhibit the entry of DV-1 and DV-2 into HepG2 cells and C6/36 mosquito cells. As such, the DV DIII protein is indeed important for the interaction with cellular receptors in both human and mosquito cells. In addition, this protein induced antibodies that completely neutralized homologous dengue serotypes although not with the same efficiency among the heterologous serotypes. This observation may be of importance when formulating a generic vaccine that is effective against all dengue virus serotypes.     

Bovine papillomavirus E1 protein is sumoylated by the host cell Ubc9 protein

Papillomavirus E1 protein is the replication initiator that recognizes and binds to the viral origin and initiates DNA strand separation through its ATP-dependent helicase activity. The E1 protein also functions in viral DNA replication by recruiting several cellular proteins to the origin, including host DNA polymerase alpha and replication protein A. To identify other cellular proteins that interact with bovine papillomavirus E1, an HeLa cDNA library was screened using a yeast two-hybrid assay. The host cell sumoylating enzyme, Ubc9, was found to interact specifically with E1 both in vitro and in vivo. Mapping studies localized critical E1 sequences for interaction to amino acids 315-459 and strongly implicated leucine 420 as critical for E1.Ubc9 complex formation. In addition to binding E1, Ubc9 catalyzed the covalent linkage of the ubiquitin-like protein, SUMO-1, to E1. An E1 mutant unable to bind Ubc9 showed normal intracellular stability, but was impaired for intranuclear distribution. Failure to accumulate in appropriate nuclear subdomains may account for the previously demonstrated replication defect of a human papillomavirus 16 E1 protein that was also unable to bind Ubc9 and suggests that sumoylation is a functionally important modification with regulatory implications for papillomavirus replication.     

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.     

Novel binding between pre-membrane protein and vacuolar ATPase is required for efficient dengue virus secretion

Flavivirus pre-membrane (prM) protein is important for proper folding and secretion of envelope (E) protein. However, other non-structural functions of prM protein in the context of virus life-cycle are poorly known. In this study, we aimed to elucidate if dengue virus (DV) prM protein interacts with host proteins and contributes to viral pathogenesis by screening human liver cDNA yeast-two-hybrid library. Our study identified vacuolar ATPase (V-ATPase) as a novel interacting partner of DV prM protein and aminoacid residues from 76 to 80 of prM protein are crucial to mediate V-ATPase binding. We showed that V-ATPase plays an important role in mediating low-pH dependent entry of DV. The biological significance of prM-V-ATPase interaction is also elucidated and we have shown that this association is critical to influence efficient virus egression. This study highlighted for the first time that flavivirus prM protein interacts with V-ATPase and V-ATPase mediates both entry and egression of DV.     

Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Background

Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.

Methodology/Principal Findings

We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.

Conclusions/Significance

Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.     

与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定

【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。     

Reconstitution and molecular analysis of the hRad9-hHus1-hRad1 (9-1-1) DNA damage responsive checkpoint complex

DNA damage activates cell cycle checkpoint signaling pathways that coordinate cell cycle arrest and DNA repair. Three of the proteins involved in checkpoint signaling, Rad1, Hus1, and Rad9, have been shown to interact by immunoprecipitation and yeast two-hybrid studies. However, it is not known how these proteins interact and assemble into a complex. In the present study we demonstrated that in human cells all the hRad9 and hHus1 and approximately one-half of the cellular pool of hRad1 interacted as a stable, biochemically discrete complex, with an apparent molecular mass of 160 kDa. This complex was reconstituted by co-expression of all three recombinant proteins in a heterologous system, and the reconstituted complex exhibited identical chromatographic behavior as the endogenous complex. Interaction studies using differentially tagged proteins demonstrated that the proteins did not self-multimerize. Rather, each protein had a binding site for the other two partners, with the N terminus of hRad9 interacting with hRad1, the N terminus of hRad1 interacting with hHus1, and the N terminus of hHus1 interacting with the C terminus of hRad9's predicted PCNA-like region. Collectively, these analyses suggest a model of how these three proteins assemble to form a functional checkpoint complex, which we dubbed the 9-1-1 complex.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.     

Cyclin K inhibits HIV-1 gene expression and replication by interfering with cyclin-dependent kinase 9 (CDK9)-cyclin T1 interaction in Nef-dependent manner

Human immunodeficiency virus-1 (HIV-1) exploits a number of host cellular factors for successful survival and propagation. The viral protein Nef plays an important role in HIV-1 pathogenesis by interacting with various cellular proteins. In the present work, we identified Cyclin K (CycK) as a novel Nef-interacting protein, and for the first time, we showed that CycK inhibits HIV-1 gene expression and replication in a Nef-dependent manner. The positive elongation factor b complex comprising cyclin-dependent kinase 9 (CDK9) and Cyclin T1 is a critical cellular complex required for viral gene expression and replication. Enhanced expression of CycK in the presence of Nef induced CycK-CDK9 binding, which prevented CDK9-Cyclin T1 complex formation and nuclear translocation of CDK9, resulting in inhibition of HIV-1 long terminal repeat-driven gene expression. Furthermore, this effect of CycK was not observed with Nef-deleted virus, indicating the importance of Nef in this phenomenon. Finally, silencing of CycK in HIV-1-infected cells resulted in increased translocation of CDK9 into the nucleus, leading to increased viral gene expression and replication. These data also suggest that endogenous CycK might act as an inhibitory factor for HIV-1 gene expression and replication in T-cells. Thus, our results clearly demonstrate that CycK utilizes HIV-1 Nef protein to displace CycT1 from the positive elongation factor b complex, resulting in inhibition of HIV-1 gene expression and replication.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

U(L)31 and U(L)34 proteins of herpes simplex virus type 1 form a complex that accumulates at the nuclear rim and is required for envelopment of nucleocapsids

The herpes simplex virus type 1 (HSV-1) U(L)34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the U(L)31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with U(L)34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing U(L)31 protein fused to glutathione-S-transferase (U(L)31-GST) and U(L)34 protein fused to GST (U(L)34-GST) were demonstrated to specifically recognize the U(L)31 and U(L)34 proteins of approximately 34,000 and 30,000 Da, respectively. The U(L)31 and U(L)34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. U(L)34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of U(L)31 protein at the nuclear rim required the presence of U(L)34 protein, inasmuch as cells infected with a U(L)34 null mutant virus contained U(L)31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of U(L)34 protein exclusively at the nuclear rim required the presence of the U(L)31 gene product, inasmuch as U(L)34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a U(L)31 null virus. When transiently expressed in the absence of other viral factors, U(L)31 protein localized diffusely in the nucleoplasm, whereas U(L)34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the U(L)31 and U(L)34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the U(S)3-encoded protein kinase, previously shown to phosphorylate the U(L)34 gene product, U(L)31 and U(L)34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, U(S)3 kinase is required for even distribution of U(L)31 and U(L)34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the U(L)31 and U(L)34 deletion mutants, these data strongly suggest that the U(L)31 and U(L)34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane.     

Ubc9 mediates nuclear localization and growth suppression of BRCA1 and BRCA1a proteins

BRCA1 gene mutations are responsible for hereditary breast and ovarian cancers. In sporadic breast tumors, BRCA1 dysfunction or aberrant subcellular localization is thought to be common. BRCA1 is a nuclear-cytoplasm shuttling protein and the reason for cytoplasmic localization of BRCA1 in young breast cancer patients is not yet known. We have previously reported BRCA1 proteins unlike K109R and cancer-predisposing mutant C61G to bind Ubc9 and modulate ER-α turnover. In the present study, we have examined the consequences of altered Ubc9 binding and knockdown on the subcellular localization and growth inhibitory function of BRCA1 proteins. Our results using live imaging of YFP, GFP, RFP-tagged BRCA1, BRCA1a and BRCA1b proteins show enhanced cytoplasmic localization of K109 R and C61G mutant BRCA1 proteins in normal and cancer cells. Furthermore, down-regulation of Ubc9 in MCF-7 cells using Ubc9 siRNA resulted in enhanced cytoplasmic localization of BRCA1 protein and exclusive cytoplasmic retention of BRCA1a and BRCA1b proteins. These mutant BRCA1 proteins were transforming and impaired in their capacity to inhibit growth of MCF-7 and CAL51 breast cancer cells. Interestingly, cytoplasmic BRCA1a mutants showed more clonogenicity in soft agar and higher levels of expression of Ubc9 than parental MCF7 cells. This is the first report demonstrating the physiological link between cytoplasmic mislocalization of mutant BRCA1 proteins, loss of ER-α repression, loss of ubiquitin ligase activity and loss of growth suppression of BRCA1 proteins. Thus, binding of BRCA1 proteins to nuclear chaperone Ubc9 provides a novel mechanism for nuclear import and control of tumor growth.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification

SUMO-1 is an ubiquitin-related protein that is covalently conjugated to a diverse assortment of proteins. The consequences of SUMO-1 modification include the regulation of protein-protein interactions, protein-DNA interactions, and protein subcellular localization. At present, very little is understood about the specific mechanisms that govern the recognition of proteins as substrates for SUMO-1 modification. However, many of the proteins that are modified by SUMO-1 interact directly with the SUMO-1 conjugating enzyme, Ubc9. These interactions suggest that Ubc9 binding may play an important role in substrate recognition as well as in substrate modification. The SUMO-1 consensus sequence (SUMO-1-CS) is a motif of conserved residues surrounding the modified lysine residue of most SUMO-1 substrates. This motif conforms to the sequence "PsiKXE," where Psi is a large hydrophobic residue, K is the lysine to which SUMO-1 is conjugated, X is any amino acid, and E is glutamic acid. In this study, we demonstrate that the SUMO-1-CS is a major determinant of Ubc9 binding and SUMO-1 modification. Mutating residues in the SUMO-1-CS abolishes both Ubc9 binding and substrate modification. These findings have important implications for how SUMO-1 substrates are recognized and for how SUMO-1 is ultimately transferred to specific lysine residues on these substrates.