W. Xu - Academia.edu (original) (raw)

Papers by W. Xu

Research paper thumbnail of Conserved structure/function of the orthoreovirus major core proteins

Virus Research, 2009

Orthoreoviruses are infectious agents with genomes of 10 segments of double-stranded RNA. Detaile... more Orthoreoviruses are infectious agents with genomes of 10 segments of double-stranded RNA. Detailed molecular information is available for all 10 segments of several mammalian orthoreoviruses, and for most segments of several avian orthoreoviruses (ARV). We, and others, have reported sequences of the L2, all S-class, and all M-class genome segments of two different avian reoviruses, strains ARV138 and ARV176. We here determined L1 and L3 genome segment nucleotide sequences for both strains to complete full genome characterization of this orthoreovirus subgroup. ARV L1 segments were 3958 nucleotides long and encode lambda A major core shell proteins of 1293 residues. L3 segments were 3907 nucleotides long and encode lambda C core turret proteins of 1285 residues. These newly determined ARV segments were aligned with all currently available homologous mammalian reovirus (MRV) and aquareovirus (AqRV) genome segments. Identical and conserved amino acid residues amongst these diverse groups were mapped into known mammalian reovirus lambda 1 core shell and lambda 2 core turret proteins to predict conserved structure/function domains. Most identical and conserved residues were located near predicted catalytic domains in the lambda-class guanylyltransferase, and forming patches that traverse the lambda-class core shell, which may contribute to the unusual RNA transcription processes in this group of viruses.

Research paper thumbnail of Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

Virology Journal, 2008

The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded R... more The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp) enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses.

Research paper thumbnail of Assignment of avian reovirus temperature-sensitive mutant recombination groups B, C, and D to genome segments

Virology, 2005

Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiqui... more Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiquitous in commercial poultry and frequently isolated from acutely infected chickens. We previously described isolation of ARV temperature-sensitive (ts) mutants after nitrosoguanidine mutagenesis of wild-type ARV138, their assignment to 7 recombination groups (A-G), and genetic mapping of mutants in groups A-D to specific gene segments. For this study, wild-type serotype ARV176 was crossed with ts mutants tsE158 (Group E), tsF206 (Group F), or tsG247 (Group G) and reassortant progenies analyzed. Reassortant temperature-sensitivities were determined by efficiency of plating at permissive and non-permissive temperatures. Mapping results indicated tsE158, tsF206, and tsG247 mapped to the L1, S4, and L3 genes, respectively, which encode the λA core shell, σNS non-structural, and λC core spike proteins, respectively. Specific amino acid substitutions in each mutant were determined and locations of structural protein alterations were placed within the 3-dimensional structure of homologous mammalian reovirus proteins. Mapping recombination groups E-G marks completion of gene assignments for all seven ts mutant groups previously generated.

Research paper thumbnail of Avian Reovirus Temperature-Sensitive Mutant tsA12 Has a Lesion in Major Core Protein  A and Is Defective in Assembly

Journal of Virology, 2004

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temper... more Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperaturesensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced ϳ100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced ϳ100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein A. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the A protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu 158 substitution in ARV A lies directly under the outer face of the A protein. This may cause a perturbation in A such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.

Research paper thumbnail of Generation and characterization of a new panel of broadly reactive anti-NS1 mAbs for detection of influenza A virus

Journal of General Virology, 2013

Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for v... more Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for virus replication and may be a good target for IAV diagnosis. To generate broadly cross-reactive NS1-specific mAbs, mice were immunized with A/Hong Kong/1/1968 (H3N2) 6×His-tagged NS1 and hybridomas were screened with glutathione S-transferase-conjugated NS1 of A/Puerto Rico/8/1934 (H1N1). mAbs were isotyped and numerous IgG-type clones were characterized further. Most clones specifically recognized NS1 from various H1N1 and H3N2 IAV types by both immunoblot and immunofluorescence microscopy in mouse M1, canine Madin-Darby canine kidney and human A549 cells. mAb epitopes were mapped by overlapping peptides and selective reactivity to the newly described viral NS3 protein. These mAbs detected NS1 in both the cytoplasm and nucleus by immunostaining, and some detected NS1 as early as 5 h post-infection, suggesting their potential diagnostic use for tracking productive IAV replication and characterizing NS1 structure and function. It was also demonstrated that the newly identified NS3 protein is localized in the cytoplasm to high levels.

Research paper thumbnail of The Actin-depolymerizing Factor Homology and Charged/Helical Domains of Drebrin and mAbp1 Direct Membrane Binding and Localization via Distinct Interactions with Actin

Journal of Biological Chemistry, 2006

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosyla... more Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/ helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actinbinding proteins and, thus, carry out multiple different functions within cells. . 2 The abbreviations used are: ARF, ADP-ribosylation factor; ADFH, actin depolymerizing factor homology; GST, glutathione S-binding protein; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; mAbp1, mammalian Abp1; GFP, green fluorescent protein.

Research paper thumbnail of Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

Journal of Virology, 2010

Because they are obligate intracellular parasites, all viruses are exclusively and intimately dep... more Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular "transcriptome." Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules.

Research paper thumbnail of Conserved structure/function of the orthoreovirus major core proteins

Virus Research, 2009

Orthoreoviruses are infectious agents with genomes of 10 segments of double-stranded RNA. Detaile... more Orthoreoviruses are infectious agents with genomes of 10 segments of double-stranded RNA. Detailed molecular information is available for all 10 segments of several mammalian orthoreoviruses, and for most segments of several avian orthoreoviruses (ARV). We, and others, have reported sequences of the L2, all S-class, and all M-class genome segments of two different avian reoviruses, strains ARV138 and ARV176. We here determined L1 and L3 genome segment nucleotide sequences for both strains to complete full genome characterization of this orthoreovirus subgroup. ARV L1 segments were 3958 nucleotides long and encode lambda A major core shell proteins of 1293 residues. L3 segments were 3907 nucleotides long and encode lambda C core turret proteins of 1285 residues. These newly determined ARV segments were aligned with all currently available homologous mammalian reovirus (MRV) and aquareovirus (AqRV) genome segments. Identical and conserved amino acid residues amongst these diverse groups were mapped into known mammalian reovirus lambda 1 core shell and lambda 2 core turret proteins to predict conserved structure/function domains. Most identical and conserved residues were located near predicted catalytic domains in the lambda-class guanylyltransferase, and forming patches that traverse the lambda-class core shell, which may contribute to the unusual RNA transcription processes in this group of viruses.

Research paper thumbnail of Avian reovirus L2 genome segment sequences and predicted structure/function of the encoded RNA-dependent RNA polymerase protein

Virology Journal, 2008

The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded R... more The orthoreoviruses are infectious agents that possess a genome comprised of 10 double-stranded RNA segments encased in two concentric protein capsids. Like virtually all RNA viruses, an RNA-dependent RNA polymerase (RdRp) enzyme is required for viral propagation. RdRp sequences have been determined for the prototype mammalian orthoreoviruses and for several other closely-related reoviruses, including aquareoviruses, but have not yet been reported for any avian orthoreoviruses.

Research paper thumbnail of Assignment of avian reovirus temperature-sensitive mutant recombination groups B, C, and D to genome segments

Virology, 2005

Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiqui... more Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiquitous in commercial poultry and frequently isolated from acutely infected chickens. We previously described isolation of ARV temperature-sensitive (ts) mutants after nitrosoguanidine mutagenesis of wild-type ARV138, their assignment to 7 recombination groups (A-G), and genetic mapping of mutants in groups A-D to specific gene segments. For this study, wild-type serotype ARV176 was crossed with ts mutants tsE158 (Group E), tsF206 (Group F), or tsG247 (Group G) and reassortant progenies analyzed. Reassortant temperature-sensitivities were determined by efficiency of plating at permissive and non-permissive temperatures. Mapping results indicated tsE158, tsF206, and tsG247 mapped to the L1, S4, and L3 genes, respectively, which encode the λA core shell, σNS non-structural, and λC core spike proteins, respectively. Specific amino acid substitutions in each mutant were determined and locations of structural protein alterations were placed within the 3-dimensional structure of homologous mammalian reovirus proteins. Mapping recombination groups E-G marks completion of gene assignments for all seven ts mutant groups previously generated.

Research paper thumbnail of Avian Reovirus Temperature-Sensitive Mutant tsA12 Has a Lesion in Major Core Protein  A and Is Defective in Assembly

Journal of Virology, 2004

Members of our laboratory previously generated and described a set of avian reovirus (ARV) temper... more Members of our laboratory previously generated and described a set of avian reovirus (ARV) temperaturesensitive (ts) mutants and assigned 11 of them to 7 of the 10 expected recombination groups, named A through G (M. Patrick, R. Duncan, and K. M. Coombs, Virology 284:113-122, 2001). This report presents a more detailed analysis of two of these mutants (tsA12 and tsA146), which were previously assigned to recombination group A. The capacities of tsA12 and tsA146 to replicate at a variety of temperatures were determined. Morphological analyses indicated that cells infected with tsA12 at a nonpermissive temperature produced ϳ100-fold fewer particles than cells infected at a permissive temperature and accumulated core particles. Cells infected with tsA146 at a nonpermissive temperature also produced ϳ100-fold fewer particles, a larger proportion of which were intact virions. We crossed tsA12 with ARV strain 176 to generate reassortant clones and used them to map the temperature-sensitive lesion in tsA12 to the S2 gene. S2 encodes the major core protein A. Sequence analysis of the tsA12 S2 gene showed a single alteration, a cytosine-to-uracil transition, at nucleotide position 488. This alteration leads to a predicted amino acid change from proline to leucine at amino acid position 158 in the A protein. An analysis of the core crystal structure of the closely related mammalian reovirus suggested that the Leu 158 substitution in ARV A lies directly under the outer face of the A protein. This may cause a perturbation in A such that outer capsid proteins are incapable of condensing onto nascent cores. Thus, the ARV tsA12 mutant represents a novel assembly-defective orthoreovirus clone that may prove useful for delineating virus assembly.

Research paper thumbnail of Generation and characterization of a new panel of broadly reactive anti-NS1 mAbs for detection of influenza A virus

Journal of General Virology, 2013

Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for v... more Influenza A virus (IAV) non-structural protein 1 (NS1) has multiple functions, is essential for virus replication and may be a good target for IAV diagnosis. To generate broadly cross-reactive NS1-specific mAbs, mice were immunized with A/Hong Kong/1/1968 (H3N2) 6×His-tagged NS1 and hybridomas were screened with glutathione S-transferase-conjugated NS1 of A/Puerto Rico/8/1934 (H1N1). mAbs were isotyped and numerous IgG-type clones were characterized further. Most clones specifically recognized NS1 from various H1N1 and H3N2 IAV types by both immunoblot and immunofluorescence microscopy in mouse M1, canine Madin-Darby canine kidney and human A549 cells. mAb epitopes were mapped by overlapping peptides and selective reactivity to the newly described viral NS3 protein. These mAbs detected NS1 in both the cytoplasm and nucleus by immunostaining, and some detected NS1 as early as 5 h post-infection, suggesting their potential diagnostic use for tracking productive IAV replication and characterizing NS1 structure and function. It was also demonstrated that the newly identified NS3 protein is localized in the cytoplasm to high levels.

Research paper thumbnail of The Actin-depolymerizing Factor Homology and Charged/Helical Domains of Drebrin and mAbp1 Direct Membrane Binding and Localization via Distinct Interactions with Actin

Journal of Biological Chemistry, 2006

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosyla... more Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/ helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actinbinding proteins and, thus, carry out multiple different functions within cells. . 2 The abbreviations used are: ARF, ADP-ribosylation factor; ADFH, actin depolymerizing factor homology; GST, glutathione S-binding protein; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; mAbp1, mammalian Abp1; GFP, green fluorescent protein.

Research paper thumbnail of Quantitative Proteomic Analyses of Influenza Virus-Infected Cultured Human Lung Cells

Journal of Virology, 2010

Because they are obligate intracellular parasites, all viruses are exclusively and intimately dep... more Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular "transcriptome." Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules.