Role of Growth Hormone Receptor in HL-60 Cell Survival

Although it is presently well established that locally produced growth hormone (GH) plays a major role in the regulation of survival mechanisms in hemopoietic cells, the responsible mechanisms are poorly understood, and the involvement of the GH receptor (GHR) has not even been demonstrated to date. In this work we investigated the presence of GHR in the human promyelocytic leukemia cell line HL-60, as well as the ability of GH treatment to stimulate both GHR and survival signaling pathways downstream GHR. Our results demonstrate that (1) both GHR mRNA and GHR immunoreactivity are present in HL-60 cells; (2) GH treatment results in an increase in the phosphorylation of the GHR-associated Jak2 and Stat3 proteins, indicating the ability of the hormone to induce receptor activation; and (3) activation of GHR increases the activity of Akt, a serine/threonine kinase that plays a prominent role in the regulation of cell survival. Taken together, these results demonstrate that GHR activation promotes survival of HL-60 cells, thus suggesting that GH plays a major role in the regulation of cell survival in the hemopoietic system, via an autocrine/paracrine mechanism.     

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.     

The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg²⁺ deprivation, but previous work suggested that MgtC is not a Mg²⁺ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg²⁺ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg²⁺ concentration, indicating that it does not bind Mg²⁺. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg²⁺ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain.     

Functional Analysis of the Human Cytomegalovirus US28 Gene by Insertion Mutagenesis with the Green Fluorescent Protein Gene

The protein encoded by the US28 gene of human cytomegalovirus (HCMV) has homology to G protein-coupled receptors (GCR). Previous studies demonstrated that recombinant US28 protein can bind the β class of chemokines (K. Neote, D. DiGregorio, J. Y. Mak, R. Horuk, and T. J. Schall, Cell 72:415–425, 1993) and induce a rise in intracellular calcium after the binding of chemokines (J. L. Gao and P. M. Murphy, J. Biol. Chem. 269:28539–28542, 1994). In order to investigate the function of the US28 protein in virus-infected cells, a recombinant HCMV (HV5.8) was constructed, with the US28 open reading frame disrupted by the insertion of the Escherichia coli gpt gene and the gene for the green fluorescent protein. The US28 gene is not required for growth in human fibroblasts (HF). HF infected with wild-type HCMV bound RANTES at 24 h postinfection and demonstrated an intracellular calcium flux induced by RANTES. In cells infected with HV5.8, RANTES did not bind or induce a calcium flux, demonstrating that US28 is responsible for the β-chemokine binding and induced calcium signaling in HCMV-infected cells. The ability of the US28 gene to bind chemokines was shown to cause a significant reduction in the concentration of RANTES in the medium of infected cells. Northern analysis of RNA from infected cells showed that US28 is an early gene, while US27 (another GCR) is a late gene.Open reading frames (ORF) with homology to cellular seven transmembrane spanning receptors have been identified in the genomes of both beta and gamma herpesviruses (15, 37). Many cellular seven transmembrane spanning receptors have been shown to be G protein-coupled receptors (GCR) and comprise a superfamily of genes encoding the receptors for a variety of biological compounds, including neurotransmitters, hormones, odorants, and chemotactic agents. GCR link the binding of an extracellular ligand to processes within the cell by their activation of associated G proteins. G proteins can activate serine/threonine kinases, phosphatidylinositol 3-kinase, phospholipases, or Ras (9). These proteins, in turn, can stimulate mitogen-activated protein kinase or generate second messenger molecules, such as diacylglycerol and inositol triphosphate, resulting in the activation of protein kinase C and increases in intracellular Ca²⁺ levels (9). Ultimately, these processes result in the amplification of the initial signal transduced by the ligand-GCR interaction into complex cellular processes such as chemotaxis.GCR are receptors for chemokines, derived from chemotactic cytokine, a multigene family of 70- to 90-amino-acid soluble proteins that are excreted from a variety of cell types and play important roles in leukocyte trafficking and immune regulation (7). Two classes of these structurally similar proteins are defined by the first two of four conserved cysteines. In the α class (e.g., interleukin-8 [IL-8], MGSA, and GCP-2) the first two cysteines are separated by an intervening residue (C-X-C), while in the β class (e.g., RANTES, MIP-1α, MIP-1β, and MCP-1) they are adjacent (C-C). In general, the α-chemokines attract primarily neutrophils, while β-chemokines can have activity on monocytes, lymphocytes, eosinophils, and basophils (46).The human cytomegalovirus (HCMV) US28 ORF shows approximately 33% homology to the cellular β-chemokine receptor CCR-1 (35). Conserved features of viral and cellular proteins include the putative seven-membrane spanning regions and cysteines implicated in disulfide bond formation. The sequence homology between US28 and cellular GCR led to the identification of β-chemokines as the ligand for the viral receptor. Recombinant HCMV US28 protein expressed in 293 cells was shown to bind β-chemokines (35), and as with the binding of chemokines by their cellular receptors, the binding of ligand by recombinant US28 expressed in K562 cells led to an increase in intracellular calcium (21).During an acute infection, HCMV can be found in the blood as well as in numerous tissues, with the lungs, kidneys, salivary gland, and liver being commonly involved. HCMV has been identified in a wide variety of cells both in culture and in patients’ tissue, including epithelial cells, endothelial cells, fibroblasts, monocytes/macrophages, and lymphocytes (33, 47, 52). Because HCMV can infect cell types that respond to chemokines and cell types that produce chemokines, the viral GCR may mimic the functions of cellular GCR, but the role of the expression of a viral GCR in viral biology and the cell type in which it is important are not known.While researchers examined US28 function with recombinant protein in previous studies (21, 36), we have investigated the functions of the US28 gene expressed from the viral genome. We have constructed a recombinant HCMV with the US28 ORF disrupted by the genes for the green fluorescent protein (GFP) and guanine phosphoribosyl transferase (GPT) and have demonstrated that US28 is responsible for the functions of a GCR in HCMV-infected cells.     

The RGD Domain of Human Osteopontin Promotes Tumor Growth and Metastasis through Activation of Survival Pathways

Background

Human osteopontin (OPN), a known tumor associated protein, exists in different isoforms, whose function is unclear. It also possesses a RGD domain, which has been implicated in diverse function. Here, we use genetic approaches to systematically investigate the function of the RGD domain in different OPN isoforms on tumor progression and metastasis for 2 different solid tumor models.

Methodology/Principal Findings

Using isoform-specific qRT-PCR, we found that OPN-A and B were the main isoforms overexpressed in evaluated human tumors, which included 4 soft tissue sarcomas, 24 lung and 30 head and neck carcinomas. Overexpression of either OPN-A or B in two different cell types promoted local tumor growth and lung metastasis in SCID mouse xenografts. However, expression of either isoform with the RGD domain either mutated or deleted decreased tumor growth and metastasis, and resulted in increased apoptosis by TUNEL staining. In vitro, whereas mutation of the RGD domain did not affect cell-cell adhesion, soft agar growth or cell migration, it increased apoptosis under hypoxia and serum starvation. This effect could be mitigated when the RGD mutant cells were treated with condition media containing WT OPN. Mechanistically, the RGD region of OPN inhibited apoptosis by inducing NF-κB activation and FAK phosphorylation. Inhibition of NF-κB (by siRNA to the p65 subunit) or FAK activation (by a inhibitor) significantly increased apoptosis under hypoxia in WT OPN cells, but not in RGD mutant cells.

Conclusion/Significance

Unlike prior reports, our data suggest that the RGD domain of both OPN-A and B promote tumor growth and metastasis mainly by protecting cells against apoptosis under stressed conditions and not via migration or invasion. Future inhibitors directed against OPN should target multiple isoforms and should inhibit cell survival mechanisms that involve the RGD domain, FAK phosphorylation and NF-κB activation.     

Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.Human cytomegalovirus (HCMV) is a widespread betaherpesvirus that causes lifelong persistent infections with occasional reactivations. While HCMV infection is usually clinically unapparent in the immunocompetent host, it can cause severe disseminated infections under conditions of immunosuppression, with manifestations in the lung, retina, and gastrointestinal tract, among others (12). Various cell types support viral replication, including epithelial cells and endothelial cells (ECs), smooth muscle cells, fibroblasts, and cells of hematopoietic origin (13, 14, 18, 19, 25, 26, 37). Among these target cells, endothelial cells are assumed to contribute particularly to hematogenous dissemination of HCMV (24).While recent clinical HCMV isolates are characterized by this broad cell tropism, the target cell range becomes restricted during long-term propagation on fibroblasts (28, 33). The underlying mechanism for this cell culture adaptation is a modulation within the viral genes UL128, UL130, and UL131A (8, 11). These three genes have been shown to be essential for infection of granulocytes, dendritic cells, epithelial cells, and endothelial cells but are dispensable for infection of fibroblasts (1, 9, 11, 34, 35). The encoded proteins pUL128, pUL130, and pUL131A were reported to form a complex with the viral glycoproteins gH and gL that is distinct from the glycoprotein complex gCIII (gH/gL/gO) (35). Whereas poorly endotheliotropic HCMV strains bear just the gH/gL/gO complex in their envelopes, highly endotheliotropic strains bear both gCIII variants: gH/gL/gO and gH/gL/pUL128-131A. Deletion of any of the three genes UL128-131A results in loss of EC tropism (11), most likely because only a complete complex of gH/gL and pUL128, pUL130, and pUL131A can efficiently function in endocytic entry in ECs (21). However, functional sites within the proteins (e.g., mediating binding to the viral complex partners or interaction with a putative cellular receptor) have not yet been identified. One approach to search for candidate protein-protein interaction sites is charge-cluster-to-alanine (CCTA) mutagenesis. This method is based on the assumption that clusters of charged amino acids tend to be exposed in the tertiary structure of a protein and are thus likely to be sites of interaction with other proteins. Replacement of these charged amino acids by uncharged alanines should then target protein-protein interaction sites without destroying the protein backbone (5, 7). Applying this method to HCMV pUL128, we were able to identify a central core region within pUL128 essential for EC infection as well as contributing sites in the N-terminal half and the C terminus of the protein (22). We now aimed to extend the study to the scanning of UL130 by markerless mutagenesis in the context of a highly endotheliotropic HCMV BACmid, TB40-BAC4. The resulting mutant viruses were then characterized with regard to their ability to infect ECs to identify the relevant parts of the protein.With regard to the role of UL130 in EC infection by endocytosis, the C-terminal part of pUL130 was of special interest. A frameshift mutation that changes the last 11 amino acids (aa) of pUL130 is the most prominent difference between the poorly endotheliotropic HCMV strain Towne and the highly endotheliotropic strain HCMV-TB40-BAC4 in this region (8, 11, 27). Rhee and Davis have described a cell-penetrating pentapeptide (CPP) motif (PFVYLI) mediating internalization by endocytosis, which is clathrin and caveolin independent but may involve lipid rafts (17). Not only do the last five amino acids of pUL130 (PNLIV) bear a striking similarity to this motif, but also the entry of HCMV into ECs has been reported to occur by an endocytic pathway (20, 23). Thus, we hypothesized that the pentapeptide motif PNLIV in pUL130 might be involved in mediating endocytic uptake of HCMV in ECs, and if so, deletion of this motif should result in a nonendotheliotropic virus. A number of CPPs that are thought to be taken up by endocytosis have now been described, including VPMLK, PMLKE, VPTLK, KLPVM, and others (32). These CPPs all bear some similarity, but the exact amino acid sequence seems to be irrelevant. We thus hypothesized for UL130 that a scrambled mutant (PNLIV changed to PINVL) should still be able to mediate endocytosis of HCMV in ECs. To test these assumptions we generated a series of mutant viruses where the PNLIV motif was either deleted, scrambled (PNLIV changed to PINVL), or exchanged against a known CPP (PFVYLI [17]) and characterized them with regard to EC infectivity.     

敲减人HPIP基因表达抑制细胞生长增殖

为了探讨人造血相关的PBX相互作用蛋白质基因（HPIP）在肿瘤发生发展中的生物学作用,构建了HPIP小干扰RNA(siRNA)的真核表达载体,验证其敲减效果并观察其对细胞生长增殖的影响.根据人HPIP的cDNA序列,设计了含有小发卡结构的寡核苷酸序列,将其克隆到siRNA表达载体上;将重组质粒转染人胚肾293T细胞,通过实时定量RT-PCR及Western 印迹分析检测HPIP基因的表达水平;结晶紫实验及流式细胞技术检测敲减HPIP基因表达对细胞生长和增殖的影响,软琼脂实验检测对肿瘤细胞非锚定依赖性生长的影响.结果显示,构建的siRNA能够有效抑制HPIP基因的表达;结晶紫实验与细胞周期分析实验显示,siRNA介导的HPIP表达沉默导致细胞生长增殖的显著抑制,软琼脂实验结果表明,稳定转染HPIP siRNA能够抑制肿瘤细胞的锚定非依赖性生长.上述结果初步表明,HPIP siRNA能明显抑制肿瘤细胞的生长与增殖,可能是一个潜在的肿瘤治疗新靶点.     

The Gene Product of Human Cytomegalovirus Open Reading Frame UL56 Binds the pac Motif and Has Specific Nuclease Activity

Using the cis-acting human cytomegalovirus (HCMV) packaging elements (pac 1 and pac 2) as DNA probes, specific DNA-protein complexes were detected by electrophoretic mobility shift assay (EMSA) in both HCMV-infected cell nuclear extracts and recombinant baculovirus-infected cell extracts containing the HCMV p130 (pUL56) protein. DNA-binding proteins, which were common in uninfected and infected cell extracts, were also detected. Mutational analysis showed that only the AT-rich core sequences in these cis-acting motifs, 5′-TAAAAA-3′ (pac 1) and 5′-TTTTAT-3′ (pac 2), were required for specific DNA-protein complex formation. The specificity of the DNA-protein complexes was confirmed by EMSA competition. Furthermore, a specific endonuclease activity was found to be associated with lysates of baculovirus-infected cells expressing recombinant p130 (rp130). This nuclease activity was time dependent, related to the amount of rp130 in the assay, and ATP independent. Nuclease activity remained associated with rp130 after partial purification by sucrose gradient centrifugation, suggesting that this activity is a property of HCMV p130. We propose a possible involvement of p130 in HCMV DNA packaging.Human cytomegalovirus (HCMV), one of eight human herpesviruses, can cause serious illness in neonates as well as in immunocompromised adults (2). For example, transplant and AIDS patients may develop life-threatening diseases as a consequence of primary infection or reactivation of latent infection. Present therapeutic approaches are limited, and new strategies that may result from a better understanding of the molecular events involved in viral maturation are needed.The HCMV virion consists of an envelope, an amorphous tegument, and an icosahedral nucleocapsid, which is assembled in the nuclei of infected cells. The precise molecular events of HCMV capsid assembly and subsequent DNA packaging are not well understood. It is generally accepted that viral DNA is packaged into a procapsid consisting of major capsid protein (UL86), minor capsid protein (UL85), minor capsid protein-binding protein (UL46), smallest capsid protein (UL47/48), assembly protein (UL80.5), and proteinase precursor protein (UL80a) (8). The assembly protein is removed during DNA insertion. It is unclear how the concatenated viral DNA contacts empty capsids and is cleaved and packaged into the capsid.Recent studies with herpes simplex virus type 1 (HSV-1) mutants that were temperature sensitive suggest that cleavage of the concatenated DNA does not occur in the absence of packaging (1). One possible model would be the involvement of cleavage packaging protein(s) which could facilitate incorporation of DNA into the procapsid by attaching to a specific motif within the viral genome. With HSV-1, the UL36 gene product (ICP1) and a smaller protein (possibly encoded by UL37) are part of a complex that recognizes the HSV-specific a sequence and are required for cleavage and packaging of viral DNA from concatemers (6, 7). In addition, the HSV-1 ICP 18.5 (UL28) gene product and the pseudorabies virus (PrV) homolog (16) were also reported to play an important role in DNA packaging (1, 14). Addison et al. (1) demonstrated that empty capsids were observed under conditions nonpermissive for the expression of the HSV-1 ICP 18.5 gene product. The HSV-1 ICP 18.5 mutants failed to cleave concatenated viral DNA in noncomplementing cells, suggesting that cleavage and packaging require ICP 18.5. Similar results were reported by Mettenleiter et al. (14) for PrV mutant protein. These observations suggest that the HSV-1 UL36, UL37, and UL28 gene products are involved in cleavage and packaging of concatenated viral DNA.In a recent study, we identified and partially characterized the gene product of HCMV UL56 (4). The HCMV UL56 gene product of 130 kDa is the homolog of the HSV-1 UL28 gene product. It is therefore postulated that UL56 possesses properties comparable to those of HSV-1 UL28, implying an involvement in cleavage and packaging of DNA. The HCMV genomic a sequence is a short sequence located at both termini of the genome and repeated in an inverted orientation at the L-S junction. The a sequence plays a key role in replication as a cis-acting signal for cleavage and packaging of progeny viral DNA and circularization of the viral genome. The HCMV a sequence contains two conserved motifs, pac 1 and pac 2, which are required for cleavage and packaging of the viral DNA (18). Both sequence motifs are located on one side of the cleavage site. The pac 1 and pac 2 motifs have an AT-rich core flanked by a GC-rich sequence. During the initial step of viral DNA packaging, a capsid-associated protein may bind to the pac sequences and may be involved in cleavage of the viral DNA concatemer.In this study, electrophoretic mobility shift assays (EMSAs) were performed with DNA probes spanning the region of these cis-acting elements. These studies demonstrate that specific proteins from HCMV-infected nuclear extracts or baculovirus-UL56-infected cell extracts bind to the pac motifs. Using affinity-purified monospecific antibodies, we show that p130 is present in specific DNA-protein complexes containing the pac motifs of the viral genome. Furthermore, evidence is presented for a sequence-specific endonuclease activity of recombinant HCMV p130, using circular plasmid DNA bearing the a sequence as a substrate.     

The Dimerization Function of MinC Resides in a Structurally Autonomous C-Terminal Domain

Limited proteolysis of the Escherichia coli cell division inhibitor MinC reveals that its dimerization function resides in a structurally autonomous C-terminal domain. We show that cytoplasmic MinC is poised near the monomer-dimer equilibrium and propose that it only becomes entirely dimeric once recruited to the membrane by MinD.     

Cytomegalovirus Survival and Transferability and the Effectiveness of Common Hand-Washing Agents against Cytomegalovirus on Live Human Hands

Congenital cytomegalovirus (CMV) transmission can occur when women acquire CMV while pregnant. Infection control guidelines may reduce risk for transmission. We studied the duration of CMV survival after application of bacteria to the hands and after transfer from the hands to surfaces and the effectiveness of cleansing with water, regular and antibacterial soaps, sanitizer, and diaper wipes. Experiments used CMV AD169 in saliva at initial titers of 1 × 10⁵ infectious particles/ml. Samples from hands or surfaces (points between 0 and 15 min) were placed in culture and observed for at least 2 weeks. Samples were also tested using CMV real-time PCR. After application of bacteria to the hands, viable CMV was recovered from 17/20 swabs at 0 min, 18/20 swabs at 1 min, 5/20 swabs at 5 min, and 4/20 swabs at 15 min. After transfer, duration of survival was at least 15 min on plastic (1/2 swabs), 5 min on crackers and glass (3/4 swabs), and 1 min or less on metal and cloth (3/4 swabs); no viable virus was collected from wood, rubber, or hands. After cleansing, no viable virus was recovered using water (0/22), plain soap (0/20), antibacterial soap (0/20), or sanitizer (0/22). Viable CMV was recovered from 4/20 hands 10 min after diaper wipe cleansing. CMV remains viable on hands for sufficient times to allow transmission. CMV may be transferred to surfaces with reduced viability. Hand-cleansing methods were effective at eliminating viable CMV from hands.     

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA_C) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA_C. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA_C consists of at least three subdomains, a flexible linker, FlhA_CN, and FlhA_CC, and that FlhA_CN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA_C not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.The bacterial flagellum, which is responsible for motility, is a supramolecular complex of about 30 different proteins, and it consists of at least three substructures: the basal body, the hook, and the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. Many of the flagellar component proteins are translocated into the central channel of the growing flagellar structure and then to the distal end of the structure for self-assembly by the flagellar type III protein export apparatus (11, 16, 22). This export apparatus consists of six integral membrane proteins, FlhA, FlhB, FliO, FliP, FliQ, and FliR, and three soluble proteins, FliH, FliI, and FliJ (18, 21). These protein components show significant sequence and functional similarities to those of the type III secretion systems of pathogenic bacteria, which directly inject virulence factors into their host cells (11, 16).FliI is an ATPase (4) and forms an FliH₂-FliI complex with its regulator, FliH, in the cytoplasm (20). FliI self-assembles into a homo-hexamer and hence exhibits full ATPase activity (1, 8, 17). FliH and FliI, together with FliJ and the export substrate, bind to the export core complex, which is composed of the six integral membrane proteins, to recruit export substrates from the cytoplasm to the core complex (14) and facilitate the initial entry of export substrates into the export gate (23). FliJ not only prevents premature aggregation of export substrates in the cytoplasm (13) but also plays an important role in the escort mechanism for cycling export chaperones during flagellar assembly (3). The export core complex is believed to be located in the central pore of the basal body MS ring (11, 16, 22). In fact, it has been found that FlhA, FliP, and FliR are associated with the MS ring (5, 9). The FliR-FlhB fusion protein is partially functional, suggesting that FliR and FlhB interact with each other within the MS ring (29). The export core complex utilizes a proton motive force across the cytoplasmic membrane as the energy source to drive the successive unfolding of export substrates and their translocation into the central channel of the growing flagellum (23, 27). Here we refer to the export core complex as the “export gate,” as we have previously (8, 16, 23, 24).FlhA is a 692-amino-acid protein consisting of two regions: a hydrophobic N-terminal transmembrane region with eight predicted α-helical transmembrane spans (FlhA_TM) and a hydrophilic C-terminal cytoplasmic region (FlhA_C) (12, 15). FlhA_TM is responsible for the association with the MS ring (9). FlhA_C interacts with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB (6, 12, 21, 24) and plays a role in the initial export process with these proteins (28). It has been shown that the V404M mutation in FlhA_C increases not only the probability of FliI binding to the export gate in the absence of FliH (14) but also the efficiency of substrate translocation through the export gate in the absence of FliH and FliI (23). Recently, it has been shown that FlhA_C is also required for substrate recognition (7). These observations suggest that an interaction between FlhA_C and FliI is coupled with substrate entry, although it is not clear how.In order to understand the mechanism of substrate entry into the export gate, we characterized a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, whose mutation blocks the flagellar protein export process at 42°C (28). We show here that this mutation severely inhibits translocation of flagellar proteins through the export gate after the FliH-FliI-FliJ complex binds to the FlhA-FlhB platform of the gate and that the impaired ability of the flhA(G368C) mutant to export flagellar proteins is restored almost to wild-type levels by intragenic second-site mutations that may alter the interactions between subdomains of FlhA_C for possible rearrangement for the export function.     

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Background

Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5′ mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.

Methodology/Principal Findings

To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.

Conclusions/Significance

While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells.     

Platelet-Derived Growth Factor Is an Autocrine Stimulator for the Growth and Survival of Human Esophageal Carcinoma Cell Lines

Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-junexpression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 × 10⁵binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses β type PDGFR (PDGFRβ) within vitroauto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.