scholarly journals The Vaccinia Virus Superoxide Dismutase-Like Protein (A45R) Is a Virion Component That Is Nonessential for Virus Replication

2001 ◽  
Vol 75 (15) ◽  
pp. 7018-7029 ◽  
Author(s):  
Fernando Almazán ◽  
David C. Tscharke ◽  
Geoffrey L. Smith
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Vaccinia Virus ◽  
Virus Replication ◽  
Metal Ion ◽  
Deletion Mutant ◽  
Core Protein ◽  
Cowpox Virus ◽  
Sod Activity ◽  
Infected Cells

ABSTRACT A characterization of the A45R gene from vaccinia virus (VV) strain Western Reserve is presented. The open reading frame is predicted to encode a 125-amino-acid protein (M r, of 13,600) with 39% amino acid identity to copper-zinc superoxide dismutase (Cu-Zn SOD). Sequencing of the A45R gene from other orthopoxviruses, here and by others, showed that the protein is highly conserved in all viruses sequenced, including 16 strains of VV, 2 strains of cowpox virus, camelpox virus, and 4 strains of variola virus. In all cases the protein lacks key residues involved in metal ion binding that are important for the catalytic activity. The A45R protein was expressed inEscherichia coli, purified, and tested for SOD activity, but neither enzymatic nor inhibitory SOD activity was detected. Additionally, no virus-encoded SOD activity was detected in infected cells or purified virions. A monoclonal antibody raised against the A45R protein expressed in E. coli identified the A45R gene product as a 13.5-kDa protein that is expressed late during VV infection. Confocal microscopy of VV-infected cells indicated that the A45R protein accumulated predominantly in cytoplasmic viral factories. Electron microscopy and biochemical analyses showed that the A45R protein is incorporated into the virion core. A deletion mutant lacking the majority of the A45R gene and a revertant virus in which the deleted gene was restored were constructed and characterized. The growth properties of the deletion mutant virus were indistinguishable from those of wild-type and revertant viruses in all cell lines tested, including macrophages. Additionally, the virulence and pathogenicity of the three viruses were also comparable in murine and rabbit models of infection. A45R is unusual in being the first VV core protein described that affects neither virus replication nor virulence.

scholarly journals A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

2015 ◽  
Vol 89 (14) ◽  
pp. 7170-7186 ◽  
Author(s):  
Laurent Chatel-Chaix ◽  
Wolfgang Fischl ◽  
Pietro Scaturro ◽  
Mirko Cortese ◽  
Stephanie Kallis ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dengue Virus ◽  
Virus Replication ◽  
Strong Evidence ◽  
Drug Target ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Replication Cycle ◽  
Infected Cells ◽  
Ns3 Protease

ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

scholarly journals Vaccinia virus hemagglutinin. A novel member of the immunoglobulin superfamily.

1989 ◽  
Vol 170 (2) ◽  
pp. 571-576 ◽  
Author(s):  
D Y Jin ◽  
Z L Li ◽  
Q Jin ◽  
Y W Hao ◽  
Y D Hou
Keyword(s):  
Amino Acid ◽  
Vaccinia Virus ◽  
Functional Relationship ◽  
Immunoglobulin Superfamily ◽  
Hydrophobic Region ◽  
Acid Glycoprotein ◽  
Ig Superfamily ◽  
Infected Cells ◽  
Membrane Spanning ◽  
Human Cmv

Striking similarities between vaccinia virus hemagglutinin (VVHA) and proteins belonging to the Ig superfamily clearly indicate that VVHA, a 315-amino acid glycoprotein expressed on the surface of the infected cells, is a novel viral protein that can be added to the expanding list of the Ig superfamily. Its deduced amino acid sequence contains one Ig-like domain at the NH2 terminus, followed by two tandem repeating units and a hydrophobic region, suggestive of membrane spanning. The results offer an opportunity for the further study of the probable evolutionary and possible functional relationship between VVHA and other members of the Ig superfamily. Our observation, together with a recent finding that human CMV possibly encodes a protein similar to the MHC class I antigens (13), provides evidence supporting the fact that the viral capture of cellular Ig-related genes is more common than expected in vaccinia and other viruses, and that usage of an Ig-like domain as recognition signals might be extended from higher animals to animal viruses.

scholarly journals Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

2000 ◽  
Vol 68 (7) ◽  
pp. 3941-3948 ◽  
Author(s):  
Tong-Soo Kim ◽  
Younghun Jung ◽  
Byoung-Kuk Na ◽  
Ki-Sun Kim ◽  
Pyung-Rim Chung
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Fasciola Hepatica ◽  
Human Subjects ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Coding Region ◽  
Sod Activity ◽  
Degenerate Oligonucleotide

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

scholarly journals Characterization of a Vaccinia Virus Mutant with a Deletion of the D10R Gene Encoding a Putative Negative Regulator of Gene Expression

2006 ◽  
Vol 80 (2) ◽  
pp. 553-561 ◽  
Author(s):  
Susan Parrish ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Deletion Mutant ◽  
Negative Regulator ◽  
Wild Type Virus ◽  
Wild Type ◽  
Gene Encoding ◽  
Early Proteins ◽  
Late Transcription ◽  
Infected Cells ◽  
Virus Mutant

ABSTRACT The D9 and D10 proteins of vaccinia virus are 25% identical to each other, contain a mutT motif characteristic of nudix hydrolases, and are conserved in all sequenced poxviruses. Previous studies indicated that overexpression of D10 and, to a lesser extent, D9 decreased the levels of capped mRNAs and their translation products. Here, we further characterized the D10 protein and showed that only trace amounts are associated with purified virions and that it is expressed exclusively at late times after vaccinia virus infection. A viable deletion mutant (vΔD10) produced smaller plaques and lower virus yields than either wild-type virus or a D9R deletion mutant (vΔD9). Purified vΔD10 virions appeared normal by microscopic examination and biochemical analysis but produced 6- to 10-fold-fewer plaques at the same concentration as wild-type or vΔD9 virions. When 4 PFU per cell of wild-type or vΔD9 virions or equal numbers of vΔD10 virions were used for inoculation, nearly all cells were infected in each case, but viral early and late transcription was initiated more slowly in vΔD10-infected cells than in the others. However, viral early transcripts accumulated to higher levels in vΔD10-infected cells than in cells infected with the wild type or vΔD9. In addition, viral early and late mRNAs and cellular actin mRNA persisted longer in vΔD10-infected cells than in others. Furthermore, analysis of pulse-labeled proteins indicated prolonged synthesis of cellular and viral early proteins. These results are consistent with a role for D10 in regulating RNA levels in poxvirus-infected cells.

scholarly journals A study of the vaccinia virus interferon-γ receptor and its contribution to virus virulence

2002 ◽  
Vol 83 (8) ◽  
pp. 1953-1964 ◽  
Author(s):  
Julian A. Symons ◽  
David C. Tscharke ◽  
Nicola Price ◽  
Geoffrey L. Smith
Keyword(s):  
Vaccinia Virus ◽  
Deletion Mutant ◽  
Interferon Γ ◽  
Soluble Form ◽  
Wide Range ◽  
Skin Loss ◽  
Infected Cells ◽  
Ifn Γ ◽  
Revertant Virus ◽  
Extension Analysis

Vaccinia virus (VV) strain Western Reserve gene B8R encodes a 43 kDa glycoprotein that is secreted from infected cells early in infection as a homodimer. This protein has amino acid similarity with the extracellular domain of cellular IFN-γ receptor (IFN-γR) and binds and inhibits IFN-γ from a wide range of species. Here we demonstrate that the B8R protein also inhibits equine IFN-γ. The 5′ end of the B8R mRNA has been mapped by primer extension analysis and the contribution of IFN-γRs to VV virulence was studied by the construction of a deletion mutant lacking the B8R gene (vΔB8R) and a revertant virus (vB8R-R) in which the B8R gene was re-inserted into the deletion mutant. A recombinant virus that expressed a soluble form of the mouse IFN-γR was also constructed and studied. The virulence of these viruses was tested in rodent models of infection. In mice, the loss of the VV IFN-γR did not affect virulence compared with WT and revertant viruses, consistent with the low affinity of the VV IFN-γR for mouse IFN-γ. However, expression of the mouse soluble IFN-γR increased virus virulence slightly. In rabbit skin, loss of the VV IFN-γR produced lesions with histological differences compared with WT and revertant viruses. Lastly, the affinity constants of the VV IFN-γR for human and mouse IFN-γ were determined by surface plasmon resonance.

scholarly journals Superoxide Dismutase-Dependent, Catalase-Sensitive Peroxides in Human Endothelial Cells Infected by Rickettsia rickettsii

1998 ◽  
Vol 66 (4) ◽  
pp. 1293-1298 ◽  
Author(s):  
June E. Hong ◽  
Lisa A. Santucci ◽  
Xiaojiang Tian ◽  
David J. Silverman
Keyword(s):  
Endothelial Cells ◽  
Superoxide Dismutase ◽  
Cell Injury ◽  
Umbilical Vein ◽  
Sod Activity ◽  
Rickettsia Rickettsii ◽  
Diethyldithiocarbamic Acid ◽  
Infected Cells ◽  
Enzyme Superoxide Dismutase ◽  
Umbilical Vein Endothelial Cells

ABSTRACT The generation and intracellular accumulation of reactive oxygen species have been shown to be associated with the infection of human umbilical vein endothelial cells (HUVEC) by Rickettsia rickettsii. In response to the oxidant superoxide, the activity of the enzyme superoxide dismutase (SOD) increases following infection by this obligate intracellular bacterium. Other oxidants which are capable of oxidizing the fluorescent probe 2′,7′-dichlorofluorescin (DCFH) also accumulate intracellularly within infected cells. In the study reported here, we show that (i) an inhibitor of SOD, diethyldithiocarbamic acid, reduces the observed rise in SOD activity in infected cells by 40 to 60% and at the same time reduces the degree of intracellular oxidation of DCFH; (ii) catalase-sensitive peroxides can be detected in supernatants of R. rickettsii-infected cells shortly after rickettsial exposure; and (iii) fluorescence-activated cell sorter analysis demonstrates significant intracellular oxidant activity in infected cells within 5 h after exposure to R. rickettsii. The results of these experiments indicate that hydrogen peroxide is a major oxidant associated with infection of HUVEC by R. rickettsii and that intracellular oxidant activity sensitive to SOD inhibition is detectable early and prior to significant rickettsial multiplication and much earlier than the ultrastructural manifestations of cell injury seen by electron microscopy.

scholarly journals Oral Treatment of Cowpox and Vaccinia Virus Infections in Mice with Ether Lipid Esters of Cidofovir

2004 ◽  
Vol 48 (2) ◽  
pp. 404-412 ◽  
Author(s):  
Debra C. Quenelle ◽  
Deborah J. Collins ◽  
W. Brad Wan ◽  
James R. Beadle ◽  
Karl Y. Hostetler ◽  
...  
Keyword(s):  
Single Dose ◽  
Vaccinia Virus ◽  
Virus Replication ◽  
Oral Treatment ◽  
Ether Lipid ◽  
Cowpox Virus ◽  
Virus Infections

ABSTRACT Four newly synthesized ether lipid esters of cidofovir (CDV), hexadecyloxypropyl-CDV (HDP-CDV), octadecyloxyethyl-CDV (ODE-CDV), oleyloxypropyl-CDV (OLP-CDV), and oleyloxyethyl-CDV (OLE-CDV), were found to have enhanced activities against vaccinia virus (VV) and cowpox virus (CV) in vitro compared to those of CDV. The compounds were administered orally and were evaluated for their efficacies against lethal CV or VV infections in mice. HDP-CDV, ODE-CDV, and OLE-CDV were effective at preventing mortality from CV infection when treatments were initiated 24 h after viral inoculation, but only HDP-CDV and ODE-CDV maintained efficacy when treatments were initiated as late as 72 h postinfection. Oral pretreatment with HDP-CDV and ODE-CDV were also effective when they were given 5, 3, or 1 day prior to inoculation with CV, even when each compound was administered as a single dose. Both HDP-CDV and ODE-CDV were also effective against VV infections when they were administered orally 24 or 48 h after infection. In animals treated with HDP-CDV or ODE-CDV, the titers of both CV and VV in the liver, spleen, and kidney were reduced 3 to 7 log10. In contrast, virus replication in the lungs was not significantly reduced. These data indicate that HDP-CDV or ODE-CDV given orally is as effective as CDV given parenterally for the treatment of experimental CV and VV infections and suggest that these compounds may be useful for the treatment of orthopoxvirus infections in humans.

scholarly journals Identification and Characterization of a Second Superoxide Dismutase Gene (sodM) fromStaphylococcus aureus

2001 ◽  
Vol 183 (11) ◽  
pp. 3399-3407 ◽  
Author(s):  
Michelle Wright Valderas ◽  
Mark E. Hart
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Stationary Phases ◽  
Potassium Cyanide ◽  
Exponential Phase ◽  
Sod Activity ◽  
Protein Amino Acid ◽  
Sequence Comparisons

ABSTRACT A gene encoding superoxide dismutase (SOD), sodM, fromS. aureus was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the S. aureus SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The sodM gene expressed from a plasmid rescued an Escherichia coli double mutant (sodA sodB) under conditions that are otherwise lethal. SOD activity gels ofS. aureus RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in asodM mutant, while the two lower bands were absent in asodA mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the sodAand sodM sodA mutants but not the sodM mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the sodM sodA mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in S. aureus throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an S. aureus sodA mutant are maintained.

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