A Role for Herpesvirus Saimiri orf14 in Transformation and Persistent Infection

The product of open reading frame 14 (orf14) of herpesvirus saimiri (HVS) exhibits significant homology with mouse mammary tumor virus superantigen. orf14 encodes a 50-kDa secreted glycoprotein, as shown previously (Z. Yao, E. Maraskovsky, M. K. Spriggs, J. I. Cohen, R. J. Armitage, and M. R. Alderson, J. Immunol. 156:3260–3266, 1996). orf14 expressed from recombinant baculovirus powerfully induces proliferation of CD4-positive cells originating from several different species. To study the role of orf14 in transformation, a mutant form of HVS (HVS Δorf14) was constructed with a deletion in the orf14 gene. The transforming potential of HVS Δorf14 was tested in cell culture and in common marmosets. Parental HVS subgroup C strain 488 immortalized common marmoset T lymphocytes in vitro to interleukin-2-independent growth, while the HVS Δorf14 mutant did not produce such a growth transformation. In addition, HVS Δorf14 was nononcogenic in common marmosets. In contrast to other nononcogenic HVS mutant viruses which were repeatedly isolated from peripheral blood mononuclear cells of infected marmosets for more than 5 months, HVS Δorf14 did not persist at a high level in vivo. These results demonstrate that orf14 of HVS is not required for replication but is required for transformation and for high-level persistence in vivo.     

Role of cellular tumor necrosis factor receptor-associated factors in NF-kappaB activation and lymphocyte transformation by herpesvirus Saimiri STP

The STP oncoproteins of the herpesvirus saimiri (HVS) subgroup A strain 11 and subgroup C strain 488 are now found to be stably associated with tumor necrosis factor receptor-associated factor (TRAF) 1, 2, or 3. Mutational analyses identified residues of PXQXT/S in STP-A11 as critical for TRAF association. In addition, a somewhat divergent region of STP-C488 is critical for TRAF association. Mutational analysis also revealed that STP-C488 induced NF-kappaB activation that was correlated with its ability to associate with TRAFs. The HVS STP-C488 P10-->R mutant was deficient in human T-lymphocyte transformation to interleukin-2-independent growth but showed wild-type phenotype for marmoset T-lymphocyte transformation in vitro and in vivo. The STP-C488 P10-->R mutant was also defective in Rat-1 fibroblast transformation, and fibroblast cell transformation was blocked by a TRAF2 dominant-negative mutant. These data implicate TRAFs in STP-C488-mediated transformation of human lymphocytes and rodent fibroblasts. Other factors are implicated in immortalization of common marmoset T lymphocytes and may also be critical in the transformation of human lymphocytes and rodent fibroblasts.     

Mutation of the Lck-Binding Motif of Tip Enhances Lymphoid Cell Activation by Herpesvirus Saimiri

The proline-rich SH3-binding (SH3B) motif of the tyrosine kinase-interacting protein (Tip) of herpesvirus saimiri (HVS) is required for binding to the cellular Src family kinase Lck. We constructed a mutant form of HVS in which prolines in the SH3B motif of Tip were altered to alanines. This mutant form of Tip was incapable of binding to Lck. The mutant virus, HVS/Tip mSH3B, retained its ability to immortalize common marmoset lymphocytes in culture. In fact, common marmoset lymphocytes immortalized by the HVS/Tip mSH3B mutant displayed increased expression of HLA-DR lymphocyte activation marker, an altered pattern of tyrosine phosphorylation, increased expression of the tyrosine kinase Lyn, and a shift in electrophoretic mobility of Lck compared to cells immortalized by wild-type HVS. Experimental infection of common marmosets resulted in fulminant lymphoma with both HVS/Tip mSH3B and wild-type HVS. However, HVS/Tip mSH3B produced greater infiltration of affected organs by proliferating lymphoid cells compared to wild-type HVS. These results demonstrate that Tip binding to Lck is not necessary for transformation and that abrogation of Tip binding to Lck alters the characteristics of transformed cells and the severity of the pathologic lesions.     

Identification and characterization of the herpesvirus saimiri oncoprotein STP-C488.

The protein encoded by herpesvirus saimiri transforming gene STP-C488 was identified and characterized. Antibodies were produced in rabbits by immunization with keyhole limpet hemocyanin-conjugated synthetic peptides specific for the predicted sequence of STP-C488. STP-C488-encoded protein was detected in recombinant Escherichia coli, transformed Rat-1 cells, transfected COS-1 cells, and in common marmoset T lymphocytes immortalized by herpesvirus saimiri strain 488. STP-C488 protein was sensitive to treatment by bacterial collagenase, consistent with the 18 uninterrupted collagenlike repeats predicted by the DNA sequence. The apparent molecular size of STP-C488 in sodium dodecyl sulfate (SDS)-polyacrylamide gels (20 to 22 kDa) was considerably larger than that predicted from the DNA sequence (9.9 kDa). Using indirect immunofluorescence tests and subcellular fractionation, STP-C488 was found to be membrane bound, primarily in perinuclear compartments. The 18 uninterrupted collagenlike repeats, sensitivity to collagenase, location in the cell, and anomalous migration through SDS-polyacrylamide gels suggest an unusual, membrane-associated, fibrous structure for this transforming herpesvirus oncoprotein.     

Downregulation of p56(lck) tyrosine kinase activity in T cells of squirrel monkeys (Saimiri sciureus) correlates with the nontransforming and apathogenic properties of herpesvirus saimiri in its natural host

Herpesvirus saimiri is capable of transforming T lymphocytes of various primate species to stable growth in culture. The interaction of the T-cellular tyrosine kinase p56(lck) with the transformation-associated viral protein Tip has been shown before to activate the kinase and provides one model for the T-cell-specific transformation by herpesvirus saimiri subgroup C strains. In contrast to other primate species, squirrel monkeys (Saimiri sciureus) are naturally infected with the virus without signs of lymphoma or other disease. Although the endogenous virus was regularly recovered from peripheral blood cells from squirrel monkeys, we observed that the T cells lost the virus genomes in culture. Superinfection with virus strain C488 did not induce growth transformation, in contrast to parallel experiments with T cells of other primate species. Surprisingly, p56(lck) was enzymatically inactive in primary T-cell lines derived from different squirrel monkeys, although the T cells reacted appropriately to stimulatory signals. The cDNA sequence revealed minor point mutations only, and transfections in COS-7 cells demonstrated that the S. sciureus lck gene codes for a functional enzyme. In S. sciureus, the tyrosine kinase p56(lck) was not activated after T-cell stimulation and enzymatic activity could not be induced by Tip of herpesvirus saimiri C488. However, the suppression of p56(lck) was partially released after administration of the phosphatase inhibitor pervanadate. This argues for unique species-specific conditions in T cells of S. sciureus which may interfere with the transforming activity and pathogenicity of herpesvirus saimiri subgroup C strains in their natural host.     

Association of the viral oncoprotein STP-C488 with cellular ras.

The STP-C488 oncogene of herpesvirus saimiri has transforming activity independent of the rest of the viral genome. We now demonstrate that STP-C488 associates with cellular ras in transformed cells. Mutations that disrupted this association with ras disrupted the transforming ability of the STP-C488 oncogene. Binding assays showed that STP-C488 was capable of competing with raf-1 for binding to ras. Expression of STP-C488 activated the ras signaling pathway as evidenced by a two- to fourfold increase in the ratio of ras-GTP to ras-GDP and by the constitutive activation of mitogen-activated protein kinase. Consistent with an activation of signaling through ras, STP-C488 expression induced ras-dependent neurite outgrowth in PC12 cells. STP-C488 is the first virus-encoded protein shown to achieve oncogenic transformation via association with cellular ras.     

STP-C, an oncoprotein of herpesvirus saimiri augments the activation of NF-kappaB through ubiquitination of TRAF6

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. Previous study has shown that STP-C, an oncoprotein of HVS, activates NF-kappaB signaling pathway. However, the detailed mechanism of STP-C-mediated NF-kappaB activation has not been reported yet. We first report that STP-C interacts with TRAF6 protein in vivo and in vitro and further investigation shows that Glu(12) residue of STP-C is critical for binding to TRAF6. Introduction of ubiquitin together with STP-C augments NF-kappaB activity compared to that of STP-C expression alone. STP-C expression further induces ubiquitination of endogenous TRAF6. In addition, either a deubiquitination enzyme, CYLD or a dominant negative E2-conjugation enzyme reduced NF-kappaB activity in spite of the presence of STP-C, supporting that the interaction between STP-C and TRAF6 induces ubiquitination of TRAF6. NF-kappaB activation by STP-C through the ubiquitinated TRAF6 causes the increased production of IL-8, an inflammatory chemokine and the enhanced expression of costimulatory molecule ICAM, which might ultimately contribute cellular transformation by the exposure of HVS-infected cells with inflammatory microenvironment and chronic activation.     

Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein.

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.     

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N₂. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N₂ gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.     

Genetics and Physiology of Acetate Metabolism by the Pta-Ack Pathway of Streptococcus mutans

In the dental caries pathogen Streptococcus mutans, phosphotransacetylase (Pta) catalyzes the conversion of acetyl coenzyme A (acetyl-CoA) to acetyl phosphate (AcP), which can be converted to acetate by acetate kinase (Ack), with the concomitant generation of ATP. A ΔackA mutant displayed enhanced accumulation of AcP under aerobic conditions, whereas little or no AcP was observed in the Δpta or Δpta ΔackA mutant. The Δpta and Δpta ΔackA mutants also had diminished ATP pools compared to the size of the ATP pool for the parental or ΔackA strain. Surprisingly, when exposed to oxidative stress, the Δpta ΔackA strain appeared to regain the capacity to produce AcP, with a concurrent increase in the size of the ATP pool compared to that for the parental strain. The ΔackA and Δpta ΔackA mutants exhibited enhanced (p)ppGpp accumulation, whereas the strain lacking Pta produced less (p)ppGpp than the wild-type strain. The ΔackA and Δpta ΔackA mutants displayed global changes in gene expression, as assessed by microarrays. All strains lacking Pta, which had defects in AcP production under aerobic conditions, were impaired in their abilities to form biofilms when glucose was the growth carbohydrate. Collectively, these data demonstrate the complex regulation of the Pta-Ack pathway and critical roles for these enzymes in processes that appear to be essential for the persistence and pathogenesis of S. mutans.     

Growth transformation of human T cells by herpesvirus saimiri requires multiple Tip-Lck interaction motifs

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.     

Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE-homologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli, did not differ from that of the parent strain. When 175 μM extrinsic H₂O₂ was added to the culture medium, bacterial growth of the ΔkatE1 strain was delayed and growth of the ΔkatE1 ΔkatE2 and ΔkatE1 ΔahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1 strain to the inhibition of growth by H₂O₂ was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H₂O₂ stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ΔkatE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H₂O₂ during logarithmic growth and that the function of these genes was influenced by incubation temperature.     

Sgs1, a Homologue of the Bloom''s and Werner''s Syndrome Genes, Is Required for Maintenance of Genome Stability in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability. sgs1Δ strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1Δ mutants, although meiosis I chromosome missegregation has been shown to be elevated. sgs1Δ suppresses the slow growth of a top3Δ strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1Δ strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3Δ or a sgs1Δ strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.     

A Novel Protein,RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in the lifestyle of streptococci. Moreover, a strain D39 ΔrafX mutant was impaired in bacterial growth, autolysis, bacterial division, and morphology. We observed that a strain R6 ΔrafX mutant was reduced in adhesion relative to the wild-type R6 strain, which was supported by an inhibition assay and a reduced amount of CbpA protein on the ΔrafX mutant bacterial cell surface, as shown by flow cytometric analysis. Finally, ΔrafX mutants were significantly attenuated in virulence in a murine sepsis model. Together, these findings suggest that RafX contributes to the biosynthesis of WTA, which is essential for full pneumococcal virulence.     

Phenotypes of Campylobacter jejuni luxS Mutants Are Depending on Strain Background,Kind of Mutation and Experimental Conditions

Since the discovery that Campylobacter (C.) jejuni produces Autoinducer 2 (AI-2), various studies have been conducted to explore the function and role of AI-2 in C. jejuni. However, the interpretation of these analyses has been complicated by differences in strain backgrounds, kind of mutation and culture conditions used. Furthermore, all research on AI-2 dependent phenotypes has been conducted with AI-2 synthase (luxS) mutants. This mutation also leads to a disruption of the activated-methyl-cycle. Most studies lack sufficient complementation resulting in not knowing whether phenotypes of luxS mutants depend on disrupted metabolism or lack of AI-2. Additionally, no AI-2 receptor has been found yet. All this contributes to an intensive discussion about the exact role of AI-2 in C. jejuni. Therefore, we examined the impact of different experiment settings on three different C. jejuni luxS mutants on growth and motility (37°C and 42°C). Our study showed that differing phenotypes of C. jejuni luxS mutants depend on strain background, mutation strategy and culture conditions. Furthermore, we complemented experiments with synthetic AI-2 or homocysteine as well as the combination of both. Complementation with AI-2 and AI-2+homocysteine significantly increased the cell number of C. jejuni NCTC 11168ΔluxS in stationary phase compared to the non-complemented C. jejuni NCTC 11168ΔluxS mutant. Genetic complementation of both C. jejuni 81-176 luxS mutants resulted in wild type comparable growth curves. Also swarming ability could be partially complemented. While genetic complementation restored swarming abilities of C. jejuni 81-176ΔluxS, it did not fully restore the phenotype of C. jejuni 81-176::luxS, which indicates that compensatory mutations in other parts of the chromosome and/or potential polar effects may appear in this mutant strain. Also with neither synthetic complementation, the phenotype of the wild type-strains was achieved, suggesting yet another reason for differing phenotypes other than communication and methionine metabolism for C. jejuni luxS mutants.