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D X Liu

Publications and source records attributed to D X Liu.

At least 19 recordsLinked to original sources
PubMed2006

Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells.

The N-terminal domain of the coronavirus nucleocapsid (N) protein adopts a fold resembling a right hand with a flexible, positively charged beta-hairpin and a hydrophobic palm. This domain was shown to interact with the genomic RNA for coronavirus infectious bronchitis virus (IBV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Based on its 3D structure, we used site-directed mutagenesis to identify residues essential for the RNA-binding activity of the IBV N protein and viral infectivity. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells. In contrast, mutation of amino acid Gln-74 to an alanine, which does not affect the binding activity of the N-terminal domain, showed minimal, if any, detrimental effect on the infectivity of IBV. This study thus identifies residues critical for RNA binding on the nucleocapsid surface, and presents biochemical and genetic evidence that directly links the RNA binding capacity of the coronavirus N protein to the viral infectivity in cultured cells. This information would be useful in development of preventive and treatment approaches against coronavirus infection.

Amino Acid Sequence↗
PubMed2006

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein.

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5'-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3'-end 1.2kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5kDa. Mutagenesis studies revealed that a CUG codon ((2570)CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at (2603)AUG and (2666)AUG, respectively. Translation initiation of the p27 expression at the (2570)CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

Blotting, Western↗
PubMed2006

Biochemical evidence for the presence of mixed membrane topologies of the severe acute respiratory syndrome coronavirus envelope protein expressed in mammalian cells.

Coronavirus envelope (E) protein is a small integral membrane protein with multi-functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N- and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N-terminus exposed luminally and the C-terminus exposed cytoplasmically (N(exo(lum)-)C(cyto)). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV.

Amino Acid Sequence↗
PubMed2006

Biochemical and functional characterization of Epstein-Barr virus-encoded BARF1 protein: interaction with human hTid1 protein facilitates its maturation and secretion.

EBV BARF1 gene encodes a secretory protein with transforming and mitogenic activities. In this report, the post-translational modification, folding, maturation and secretion of BARF1 are systematically studied by site-directed mutagenesis and overexpression of the protein in mammalian cells using the vaccinia/T7 system. The protein was shown to be post-translationally modified by N-linked glycosylation on the asparagine 95 residue. This modification was confirmed to be essential for the maturation and secretion of the protein. Analysis of the four cysteine residues by site-directed mutagenesis demonstrated that cysteine 146 and 201 were essential for proper folding and secretion of the protein. To search for human proteins involved in the maturation process of the protein, a yeast two-hybrid screening was carried out using the BARF1 sequence from amino acids 21-221 (BARF1Delta) as bait, leading to the identification of human hTid1 protein as a potential interacting protein. This interaction was subsequently confirmed by coimmunoprecipitation and dual immunofluorescent labeling of cells coexpressing BARF1 and hTid1, and the interaction domain in hTid1 was mapped to amino acids 149-320. Interestingly, coexpression of BARF1 with hTid1 demonstrated that hTid1 could promote secretion of BARF1, suggesting that hTid1 may act as a chaperone to facilitate the folding, processing and maturation of BARF1.

HSP40 Heat-Shock Proteins↗
PubMed2006

Biochemical and functional characterization of the membrane association and membrane permeabilizing activity of the severe acute respiratory syndrome coronavirus envelope protein.

A diverse group of cytolytic animal viruses encodes small, hydrophobic proteins to modify host cell membrane permeability to ions and small molecules during their infection cycles. In this study, we show that expression of the SARS-CoV E protein in mammalian cells alters the membrane permeability of these cells. Immunofluorescent staining and cell fractionation studies demonstrate that this protein is an integral membrane protein. It is mainly localized to the ER and the Golgi apparatus. The protein can be translocated to the cell surface and is partially associated with lipid rafts. Further biochemical characterization of the protein reveals that it is posttranslationally modified by palmitoylation on all three cysteine residues. Systematic mutagenesis studies confirm that the membrane permeabilizing activity of the SARS-CoV E protein is associated with its transmembrane domain.

Amino Acid Sequence↗
PubMed2006

Identification of Hepta- and Octo-Uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants.

Programmed frameshifting is one of the translational recoding mechanisms that read the genetic code in alternative ways. This process is generally programmed by signals at defined locations in a specific mRNA. In this study, we report the identification of hepta- and octo-uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of severe acute respiratory syndrome coronavirus (SARS-CoV) ORF 3a variants. SARS-CoV ORF 3a encodes a minor structural protein of 274 amino acids. Over the course of cloning and expression of the gene, a mixed population of clones with six, seven, eight and nine T stretches located 14 nt downstream of the initiation codon was found. In vitro and in vivo expression of clones with six, seven and eight Ts, respectively, showed the detection of the full-length 3a protein. Mutagenesis studies led to the identification of the hepta- and octo-uridine stretches as slippery sequences for efficient frameshifting. Interestingly, no stimulatory elements were found in the sequences upstream or downstream of the slippage site. When the hepta- and octo-uridine stretches were used to replace the original slippery sequence of the SARS-CoV ORF 1a and 1b, efficient frameshift events were observed. Furthermore, the efficiencies of frameshifting mediated by the hepta- and octo-uridine stretches were not affected by mutations introduced into a downstream stem-loop structure that totally abolish the frameshift event mediated by the original slippery sequence of ORF 1a and 1b. Taken together, this study identifies the hepta- and octo-uridine stretches that function as sole elements for efficient +1 and -1 ribosomal frameshift events.

Animals↗
PubMed2005

Selection of and recombination between minor variants lead to the adaptation of an avian coronavirus to primate cells.

An interesting question posed by the current evidence that severe acute respiratory syndrome coronavirus may be originated from an animal coronavirus is how such an animal coronavirus breaks the host species barrier and becomes zoonotic. In this report, we study the chronological order of genotypic changes in the spike protein of avian coronavirus infectious bronchitis virus (IBV) during its adaptation to a primate cell line. Adaptation of the Beaudette strain of IBV from chicken embryo to Vero cells showed the accumulation of 49 amino acid mutations. Among them, 26 (53.06%) substitutions were located in the S protein. Sequencing analysis and comparison of the S gene demonstrated that the majority of the mutations were accumulated and fixed at passage 7 on Vero cells and minor variants were isolated in several passages. Evidence present suggests that the dominant Vero cell-adapted IBV strain may be derived from the chicken embryo passages by selection of and potential recombination between the minor variants. This may explain why adaptation is a rapid process and the dominant strain, once adapted to a new host cell, becomes relatively stable.

Adaptation, Physiological↗
PubMed2005

Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus.

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein of 422 amino acids. Similar to other coronavirus N proteins, SARS-CoV N protein is predicted to be phosphorylated and may contain nuclear localization signals, serine/arginine-rich motif, RNA binding domain and regions responsible for self-association and homo-oligomerization. In this study, we demonstrate that the protein is posttranslationally modified by covalent attachment to the small ubiquitin-like modifier. The major sumoylation site was mapped to the (62)lysine residue of the N protein. Further expression and characterization of wild type N protein and K62A mutant reveal that sumoylation of the N protein drastically promotes its homo-oligomerization, and plays certain roles in the N protein-mediated interference of host cell division. This is the first report showing that a coronavirus N protein undergoes posttranslational modification by sumoylation, and the functional implication of this modification in the formation of coronavirus ribouncleoprotein complex, virion assembly and virus-host interactions.

Cell Division↗
PubMed2004

Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability.

To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in Escherichia coli. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in E. coli cells.

Animals↗
PubMed2004

A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature.

The spike (S) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. A number of variants with deletions and mutations in the S protein have been isolated from naturally and persistently infected animals and tissue cultures. Here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (IBV) to a monkey kidney cell line. The complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. A single amino acid reversion (L294-to-Q) in the S protein is sufficient to abrogate the ts phenotype. Interestingly, unlike wt virus, the revertant grows well at and below 32 degrees C, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. If the two ts mutants were allowed to enter cells at 32 degrees C, the S protein was synthesized, core-glycosylated and at least partially modified at 40 degrees C. However, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 degrees C. Evidence presented demonstrated that the Q294-to-L294 mutation, located at a highly conserved domain of the S1 subunit, might hamper processing of the S protein to a matured 180-kDa, endo-glycosidase H-resistant glycoprotein and the translocation of the protein to the cell surface. Consequently, some essential functions of the S protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished.

Acclimatization↗
PubMed2004

Cell cycle molecules and vertebrate neuron death: E2F at the hub.

Vertebrate neuron cell death is both a normal developmental process and the catastrophic outcome of nervous system trauma or degenerative disorders. Although the mechanisms of such death include an evolutionarily conserved core apoptotic pathway that is highly homologous to that first described by Horvitz and co-workers in Caenorhabditis elegans, it appears that many instances of neuron death additionally require the transcription-dependent induction of proapoptotic molecules. One such proapoptotic transcriptional pathway revealed by studies over the past decade revolves about the transcription factor E2F and those molecules that either regulate E2F activity or that are direct or indirect transcriptional targets of E2F. Many of the molecules associated with the E2F apoptotic pathway in postmitotic neurons also participate in the cell cycle in proliferating cells. Observations in human material and in animal and cell culture models show widespread correlation between changes in expression, activity and subcellular localization of E2F-related cell cycle molecules and developmental and catastrophic neuron death. A variety of experimental approaches support a causal role for such changes in the death process and are beginning to indicate how the neuronal E2F pathway activates the core apoptotic machinery. The discovery and elaboration of the neuronal apoptotic E2F pathway provides abundant targets as well as small molecule candidates for potential therapeutic intervention in nervous system trauma and degenerative disease.

Animals↗
PubMed2003

Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication.

The subgenomic RNA 3 of IBV has been shown to be a tricistronic mRNA, encoding three products in IBV-infected cells. To explore if the least expressed ORF, ORF 3b, which encodes a nonstructural protein, is evolutionarily conserved and functionally indispensable for viral propagation in cultured cells, the Beaudette strain of IBV was propagated in chicken embryonated eggs for three passages and then adapted to a monkey kidney cell line, Vero. The 3b gene of passage 3 in embryonated eggs and passages 7, 15, 20, 25, 30, 35 50, and 65 in Vero cells were amplified by reverse transcription-polymerase chain reaction and sequenced. The results showed that viral RNA extracted from passages 35, 50, and 65 contained a single A insertion in a 6A stretch of the 3b gene between nucleotides 24075 and 24080, whereas the early passages carried the normal 3b gene. This insertion resulted in a frameshift event and therefore, if expressed, a C-terminally truncated protein. We showed that the frameshifting product, cloned in a plasmid, was expressed in vitro and in cells transfected with the mutant construct. The normal product of the 3b gene is 64 amino acids long, whereas the frameshifting product is 34 amino acids long with only 17 homogeneous amino acid residues at the N-terminal half. Immunofluorescent studies revealed that the normal 3b protein was localized to the nucleus and the truncated product showed a "free" distribution pattern, indicating that the C-terminal portion of 3b was responsible for its nuclear localization. Comparison of the complete genome sequences (27.6 kb) of isolates p20c22 and p36c12 (from passages 20 and 36, respectively) revealed that p36c12 contains three amino acid substitutions, two in the 195-kDa protein (encoded by gene 1) and one in the S protein, in addition to the frameshifting 3b product. Further characterization of the two isolates demonstrated that p36c12 showed growth advantage over p20c22 in both Vero cells and chicken embryos and was more virulent in chicken embryos than p20c22. These results suggest that the 3b gene product is not essential for the replication of IBV.

Adaptation, Physiological↗
PubMed2002

Membrane association and dimerization of a cysteine-rich, 16-kilodalton polypeptide released from the C-terminal region of the coronavirus infectious bronchitis virus 1a polyprotein.

More than 10 mature proteins processed from coronavirus gene 1-encoded polyproteins have been identified in virus-infected cells. Here, we report the identification of the most C-terminal cleavage product of the 1a polyprotein as a 16-kDa protein in infectious bronchitis virus-infected Vero cells. Indirect immunofluorescence demonstrated that the protein exhibits a distinct perinuclear punctate staining pattern, suggesting that it is associated with cellular membranes. Positive staining observed on nonpermeabilized cells indicates that the protein may get transported to the cell surface, but no secretion of the protein out of the cells was observed. Treatment of the membrane fraction prepared from cells expressing the 16-kDa protein with Triton X-100, a high pH, and a high concentration of salts showed that the protein may be tightly associated with intracellular membranes. Dual-labeling experiments demonstrated that the 16-kDa protein colocalized with the 5'-bromouridine 5'-triphosphate-labeled viral RNA, suggesting that it may be associated with the viral replication machinery. Sequence comparison of the 16-kDa protein with the equivalent products of other coronaviruses showed multiple conserved cysteine residues, and site-directed mutagenesis studies revealed that these conserved residues may contribute to dimerization of the 16-kDa protein. Furthermore, increased accumulation of the 16-kDa protein upon stimulation with epidermal growth factor was observed, providing preliminary evidence that the protein might be involved in the growth factor signaling pathway.

Amino Acid Sequence↗
PubMed2002

A six-nucleotide segment within the 3' untranslated region of hibiscus chlorotic ringspot virus plays an essential role in translational enhancement.

RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3' untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3' UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3' region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3' UTR.

3' Untranslated Regions↗
PubMed2001

Regulation of neuronal survival and death by E2F-dependent gene repression and derepression.

Neuronal death induced by a variety of means requires participation of the E2F family of transcription factors. Here, we show that E2F acts as a gene silencer in neurons and that repression of E2F-responsive genes is required for neuronal survival. Moreover, neuronal death evoked by DNA damaging agents or trophic factor withdrawal is characterized by derepression of E2F-responsive genes. Such derepression, rather than direct E2F-promoted gene activation, is required for death. Among the genes that are derepressed in neurons subjected to DNA damage or trophic factor withdrawal are the transcription factors B- and C-myb. Overexpression of B- and C-myb is sufficient to evoke neuronal death. These findings support a model in which E2F-dependent gene repression and derepression play pivotal roles in neuronal survival and death, respectively.

Animals↗
PubMed2001

Further identification and characterization of novel intermediate and mature cleavage products released from the ORF 1b region of the avian coronavirus infectious bronchitis virus 1a/1b polyprotein.

The coronavirus 3C-like proteinase is one of the viral proteinases responsible for processing of the 1a and 1a/1b polyproteins to multiple mature products. In cells infected with avian coronavirus infectious bronchitis virus (IBV), three proteins of 100, 39, and 35 kDa, respectively, were previously identified as mature cleavage products released from the 1b region of the 1a/1b polyprotein by the 3C-like proteinase. In this report, we show the identification of two more cleavage products of 68 and 58 kDa released from the same region of the polyprotein. In addition, two stable intermediate cleavage products with molecular masses of 160 and 132 kDa, respectively, were identified in IBV-infected cells. The 160-kDa protein was shown to be an intermediate cleavage product covering the 100- and 68-kDa proteins, and the 132-kDa protein to be an intermediate cleavage product covering the 58-, 39-, and 35-kDa proteins. Immunofluorescent staining of IBV-infected cells and cells expressing individual cleavage products showed that the 100-, 68-, and 58-kDa proteins were associated with the membranes of the endoplasmic reticulum, and the 39- and 35-kDa proteins displayed diffuse distribution patterns.

Animals↗
PubMed2001

Differential molecular assemblies underlie the dual function of Axin in modulating the WNT and JNK pathways.

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.

Axin Protein↗