Biomedical subjects

W Yuan

Publications and source records attributed to W Yuan.

At least 37 records · Page 2Linked to original sources

PubMed2001

In vitro translational analysis of genomic, defective, and satellite RNAs of Cryphonectria hypovirus 3-GH2.

Cryphonectria hypovirus 3-GH2 (CHV3-GH2) is a member of the fungal virus family Hypoviridae that differs from previously characterized members in having a single large open reading frame with the potential to encode a protein of 326 kDa from its 9.8-kb genome. The N-terminal portion of the ORF contains sequence motifs that are somewhat similar to papain-like proteinases identified in other hypoviruses. Translation of the ORF is predicted to release autocatalytically a 32.5-kDa protein. A defective RNA, predicted to encode a 91.6-kDa protein representing most of the N-terminal proteinase fused to the entire putative helicase domain, and two satellite RNAs, predicted to encode very small proteins, also are associated with CHV3-GH2 infected fungal cultures. We performed in vitro translation experiments to examine expression of these RNAs. Translation of three RT-PCR clones representing different lengths of the amino-terminal portion of the ORF of the genomic RNA resulted in autocatalytic release of the predicted 32.5-kDa protein. Site-directed mutagenesis was used to map the processing site between Gly(297) and Thr(298). In vitro translation of multiple independent cDNA clones of CHV3-GH2-defective RNA 2 resulted in protein products of approximately 92 kDa, predicted to be the full-length translation product, 32 kDa, predicted to represent the N-terminal proteinase, and 60 kDa, predicted to represent the C-terminal two-thirds of the full-length product. In vitro translation of cDNA clones representing satellite RNA 4 resulted in products of slightly less than 10 kDa, consistent with the predicted 9.4 kDa product.

Amino Acid Sequence↗

PubMed2001

Involvement of chemokine receptors in breast cancer metastasis.

Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumour cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1alpha and CCL21/6Ckine exhibit peak levels of expression in organs representing the first destinations of breast cancer metastasis. In breast cancer cells, signalling through CXCR4 or CCR7 mediates actin polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. In vivo, neutralizing the interactions of CXCL12/CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung. Malignant melanoma, which has a similar metastatic pattern as breast cancer but also a high incidence of skin metastases, shows high expression levels of CCR10 in addition to CXCR4 and CCR7. Our findings indicate that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.

Actins↗

PubMed2001

Type I error in sample size re-estimations based on observed treatment difference.

Sample size re-estimation based on an observed difference can ensure an adequate power and potentially save a large amount of time and resources in clinical trials. One of the concerns for such an approach is that it may inflate the type I error. However, such a possible inflation has not been mathematically quantified. In this paper the mathematical mechanism of this inflation is explored for two-sample normal tests. A (conditional) type I error function based on normal data is derived. This function not only provides the quantification but also gives mathematical mechanisms of possible inflation in the type I error due to the sample size re-estimation. Theoretically, based on their decision rules (certain upper and lower bounds), people can calculate this function and exactly visualize the changes in type I error. Computer simulations are performed to ensure the results. If there are no bounds for the adjustment, the inflation is evident. If proper adjusting rules are used, the inflation can be well controlled. In some cases the type I error can even be reduced. The trade-off is to give up some 'unrealistic power'. We investigated several scenarios in which the mechanisms to change the type I error are different. Our simulations show that similar results may apply to other distributions.

Clinical Trials as Topic↗

PubMed2001

Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein.

Of the several hundred proteins induced by interferon (IFN) alpha/beta, the ubiquitin-like ISG15 protein is one of the most predominant. We demonstrate the novel way in which the function of the ISG15 protein is inhibited by influenza B virus, which strongly induces the ISG15 protein: a specific region of the influenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of ISG15 to its target proteins both in vitro and in infected cells. We identify UBE1L as the E1 enzyme that catalyzes the first activation step in the conjugation of ISG15, and show that the NS1B protein inhibits this activation step in vitro. Influenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection. We discuss the likely basis for these different strategies.

Amino Acid Sequence↗

PubMed2001

Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.

Acyl Coenzyme A↗

PubMed2001

Canine parvovirus capsid assembly and differences in mammalian and insect cells.

We examined the assembly processes of the capsid proteins of canine parvovirus (CPV) in mammalian and insect cells. In CPV-infected cells empty capsids assembled within 15 min, and then continued to form over the following 1 h, while full (DNA-containing) capsids were detected only after 60 min, and those accumulated slowly over several hours. In cells expressing VP1 and VP2 or only VP2, empty capsid formation was also efficient, but was slightly slower than that in infected cells. Small amounts of trimer forms of VP2 were detected in cells expressing wild type capsid proteins, but were not seen for mutants containing changes that prevented capsid assembly. CPV capsids accumulated in the cell nucleus, but mutant VP1 and VP2 proteins that did not assemble became distributed throughout the nucleus and the cytoplasm, irrespective of whether they were expressed as VP1 and VP2, or as VP2 only. Urea or pH treatment of empty capsids released dimer, trimer, or pentamer capsid protein combinations, while treatment of full capsids consistently released trimer and, in some cases, pentamer forms. When wild type or assembly-defective VP2 genes were expressed from recombinant baculoviruses in insect cells, most of the protein was recovered as noncapsid aggregates, and only a small proportion assembled into capsids. Both the assembled capsids and the noncapsid aggregates were seen primarily in the cytoplasm of the insect cells. The VP2 expressed in insect cells that was recovered in aggregates had an isoelectric point of about pH 6.3, while that recovered from assembled capsids had a pI of about 5.2, similar to that seen for the VP2 of capsids recovered from mammalian cells.

Animals↗

PubMed2001

Direct repair of defect in lumbar spondylolysis and mild isthmic spondylolisthesis by bone grafting, with or without facet joint fusion.

Forty-six patients with lumbar spondylolysis and mild isthmic spondylolisthesis were managed with direct repair of the defect with or without facet joint fusion in the affected segment. There were 24 males and 22 females, ranging in age from 15 to 56 years (average, 38.2 years). These patients had experienced clinical symptoms due to spondylolysis for between 4 months and 20 years (average, 5.3 years). Of 46 patients, 28 had no spondylolisthesis, 11 had Meyerding grade I vertebral slippage and 7 had grade II. Direct repair of 98 defects was performed on these patients. Twenty-six patients, in whom the disc adjacent to the defect was determined as degenerative by magnetic resonance imaging (MRI), simultaneously underwent facet joint fusion; 17 in one segment and 9 in two segments. The average period of follow-up was 50 months (24-92 months). Ninety-four defects achieved bony healing. As a result, 28 patients were graded as having an excellent outcome, 15 good, and 3 fair. Bone grafting in the defects achieves union between the loose lamina and the anterior element of lumbar vertebrae, and reconstructs the anatomic structure and physiologic functions of the lumbar vertebrae. There was no significant difference in outcome between the spondylolytic/spondylolisthetic patients with non-degenerative disc, who were treated with direct repair of defect only, and those with degenerative disc, who additionally underwent a fusion procedure (P > 0.05). The present series demonstrates a satisfactory result and a high rate of bony healing of the pars defect by this operative procedure in patients with lumbar spondylolysis and mild isthmic spondylolisthesis. Preoperative assessment of the disc degeneration with MRI is of great assistance in making the protocol choice of whether to opt for fusion.

Adult↗

PubMed2001

Indicators of fetal growth and infectious disease in childhood--a birth cohort with hospitalization as outcome.

We evaluated the association between indicators of fetal growth and hospitalization with infectious disease during childhood in a cohort of 10,400 newborns. The cohort was based on children born to mothers who at about 36 weeks of gestation attended the midwife centres in Odense and Aalborg, Denmark for a routine examination. Women were recruited to the study from April 1984 to April 1987. After linkage with the National Hospital Registry, the first hospitalization with infectious disease from 6 months up to 12 years of age was identified. The cumulative incidence of hospitalization with infectious disease during follow-up was 18.9%. Preterm birth was associated with an increased risk of being hospitalized with infections during childhood (incidence rate ratio: 1.67, 95% CI: 1.33-2.10); low birth weight had a similar association, but only in preterm birth. Reduced birth length related to the head was correlated with an increased risk of hospitalization with infections. The effect of gestational age was mainly seen in the period close to the time of birth, but the children who were short at birth appeared to remain at increased risk throughout the age interval under analysis. In conclusion, the study suggests that preterm birth was the main factor underlying the association between low birth weight and the increased risk of hospitalization with infectious disease during childhood. However, it could not explain the increased risk in children who were short at birth.

Child↗

PubMed2001

Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP.

Dentinogenesis imperfecta 1 (DGI1, MIM 125490) is an autosomal dominant dental disease characterized by abnormal dentin production and mineralization. The DGI1 locus was recently refined to a 2-Mb interval on 4q21 (ref. 1). Here we study three Chinese families carrying DGI1. We find that the affected individuals of two families also presented with progressive sensorineural high-frequency hearing loss (gene DFNA39). We identified three disease-specific mutations within the dentin sialophosphoprotein gene (DSPP) in these three families. We detected a G-->A transition at the donor-splicing site of intron 3 in one family without DFNA39, a mutation predicted to result in the skipping of exon 3. In two other families affected with both DGI1 and DFNA39, however, we identified two independent nucleotide transversions in exons 2 and 3 of DSPP, respectively, that cause missense mutations of two adjacent amino-acid residues in the predicted transmembrane region of the protein. Moreover, transcripts of DSPP previously reported to be expressed specifically in teeth are also detected in the inner ear of mice. We have thus demonstrated for the first time that distinct mutations in DSPP are responsible for the clinical manifestations of DGI1 with or without DFNA39.

Asian People↗

PubMed2001

Model-based analysis of dynamics in vergence adaptation.

We previously proposed a model to study the dynamics of disparity vergence responses. This model was based on known physiology and consisted of pulse and step neural control processes feeding a linear second-order oculomotor plant. Here, we apply a slightly modified version of that model to analyze the influence of short-term adaptation on vergence dynamics. This analysis showed that, unlike normal vergence responses, adapted responses could not be accurately simulated without a delay between the step and pulse components. Through simulations of normal vergence and adapted vergence responses, we found a strong correlation between delay of the step signal and the size of the movement overshoot. This correlation suggests a strong interaction between neural process generating the pulse and step motor control signals.

Adaptation, Physiological↗

PubMed2001

Maternal prenatal lifestyle factors and infectious disease in early childhood: a follow-up study of hospitalization within a Danish birth cohort.

OBJECTIVES: To examine whether maternal prenatal lifestyle factors were associated with the risk of hospitalization with infectious disease during early childhood and whether a possible association was modified by fetal growth reduction. METHODS: The study was based on a birth cohort of 10 400 newborns whose mothers attended the midwife centers in Odense and Aalborg, Denmark, from April 1984 to April 1987 at approximately the 36th week of gestation. Information on hospitalization with infectious disease was extracted from the National Hospital Discharge Registry, and newborns were followed up to the end of 1996. RESULTS: Among 6022 children who were hospitalized at least once, 31.4% (n = 1892) were hospitalized with infectious diseases. The cumulative incidence rate of hospitalization with infections in children from the age of 6 months to 12 years was 18.9%. The incidence rate ratio in children born to mothers smoking during pregnancy was 1.24 (95% confidence interval: 1.13-1.36) compared with those of nonsmoking mothers. Mothers whose body mass index was <18 kg/m(2) were more likely to give birth to infants who were hospitalized with infectious disease (incidence rate ratio: 1.29; 95% confidence interval: 1.05-1.59). The increased risk was present in children only up to the age of 5 years. CONCLUSIONS: The study shows that maternal smoking during pregnancy and a low prepregnancy body mass index are associated with a higher risk of hospitalization with infectious disease during early childhood. These associations are independent of fetal growth indicators.

Body Mass Index↗

PubMed2001

Intravascular local gene transfer mediated by protein-coated metallic stent.

OBJECTIVE: To assess the feasibility, efficiency and selectivity of adenovirus-mediated gene transfer to local arterial wall by protein-coated metallic stent. METHODS: A replication-defective recombinant adenovirus carrying the Lac Z reporter gene for nuclear-specific beta-galactosidase (Ad-beta gal) was used in this study. The coating for metallic stent was made by immersing it in a gelatin solution containing crosslinker. The coated stents were mounted on a 4.0 or 3.0 mm percutaneous transluminal coronary angioplasty (PTCA) balloon and submersed into a high-titer Ad-beta gal viral stock (2 x 10(10) pfu/ml) for 3 min, and then implanted into the carotid arteries in 4 mini-swines and into the left anterior descending branch of the coronary artery in 2 mini-swines via 8F large lumen guiding catheters. The animals were sacrificed 7 (n = 4), 14 (n = 1) and 21 (n = 1) days after implantation, respectively. The beta-galactosidase expression was assessed by X-gal staining. RESULTS: The results showed that the expression of transgene was detected in all animal. In 1 of carotid artery with an intact intima, the beta-gal expression was limited to endothelial cells. In vessels with denuded endothelium, gene expression was found in the sub-intima, media and adventitia. The transfection efficiency of medial smooth muscle cells was 38.6%. In 2 animals sacrificed 7 days after transfection, a microscopic examination of X-gal-stained samples did not show evidence of transfection in remote organs and arterial segments adjacent to the treated arterial site. CONCLUSIONS: Adenovirus-mediated arterial gene transfer to endothelial, smooth muscle cells and adventitia by protein-coated metallic stent is feasible. The transfection efficiency is higher. The coated stent may act as a good carrier of adenovirus-mediated gene transfer and have a potential to prevent restenosis following PTCA.

Adenoviridae↗

PubMed2001

Effects of ouabain and digoxin on gene expression of sodium pump alpha-subunit isoforms in rat myocardium.

OBJECTIVE: To compare the effects of ouabain and digoxin on the gene expression of sodium pump alpha-subunit isoforms in the myocardium of rats. METHODS: Normal Sprague-Dawley (SD) rats were injected with ouabain (20 micrograms.kg-1.d-1, i.p.), digoxin (32 micrograms.kg-1.d-1, i.p.) and normal saline (NS) once a day, respectively, and indirect systolic blood pressure was recorded once a week. Six weeks later, all of the rats were killed, and sodium pump alpha 1-, alpha 2-, and alpha 3-subunit mRNA levels in the myocardium were detected with the reverse transcription polymerase chain reaction (RT-PCR) method. RESULTS: The systolic blood pressure of the rats infused with ouabain increased significantly at the end of week 6 (132.6 +/- 9.0 mm Hg vs 115.7 +/- 8.2 mm Hg, P < 0.01), while no difference in blood pressure was found between the digoxin group and the NS group. The expression of sodium pump alpha-subunit isoforms in the ventricular myocardium was regulated by either ouabain or digoxin. Both ouabain and digoxin stimulated expression of the alpha 3-isoform, whereas alpha 2 was unchanged in those two groups. alpha 1-isoform expression decreased in the ouabain group and was unchanged in the digoxin group. CONCLUSIONS: These results suggest that both ouabain and digoxin could regulate sodium pump alpha-subunit isoform expression, which might be related to the physiological roles of endogenous ouabain and might be responsible for the difference in the pharmacological and toxicological effects of ouabain and digoxin, including their effects on blood pressure.

Animals↗

PubMed2001

Recording of spared motor evoked potentials and its augmentation by 4-aminopyridine in chronic spinal cord-injured rats.

OBJECTIVE: To research the direct electrophysiological evidence of discomplete spinal cord injury (SCI) and the effect of 4-aminopyridine on it. METHODS: Motor evoked potentials (MEPs), both spinal cord recorded MEPs (scMEPs) and extracellularly recorded MEPs (exMEPs) were recorded and characterized on a T13 epidural electrode (scMEPs) and an extracellular microelectrode (exMEPs) for 10 normal rats and 40 rats with lesions of various severity (sham, 35 g.cm force (gcf), 70 gcf, 100 gcf impact injury) at the T8-T9 cord using the Allen's drop model. The incline plane and Tarlov techniques were used to assess clinical neurological function. RESULTS: MEPs in the normal rats were elicited by applying transcortical suprathreshold stimulation consisting of 3-4 early negative peaks (N1, N2, N3 and N4) followed by several late waves. The N1 and N2 peaks were largest in the anterior and ventrolateral funiculus, respectively, which was indicative of extrapyramidal pathways. The 100 gcf impact injuries and the cord transection abolished the MEP distal to the lesion, whereas the 35 gcf injuries resulted in a latency shift and amplitude decrement of the MEP peaks. Eighteen of the 20 rats with 70 gcf-injuries showed clinical paraplegia. Among them, 7 rats had neurophysiological evidence of residual conduction pathways through the lesioned cord segment, such as the presence of N1 and N2 peaks in the scMEPs or exMEPs. After 4-aminopyridine (4-AP) administrations (1 mg/kg), the amplitude of the spared exMEP increased significantly and spread more widely. CONCLUSIONS: MEPs evoked by transcortical stimulation travel mostly in the extrapyramidal tract. MEP monitoring could provide an excellent method of detecting the functional integrity of the motor tracts after SCI, and could even detect spared motor fibers after discomplete SCI. Furthermore, the use of 4-AP or other K+ channel blocking agents may be a potential treatment for patients with chronic moderate to severe SCI.

4-Aminopyridine↗

PubMed2001

[Differential regulation of sodium pump alpha-subunit isoform gene by ouabain and digoxin in myocardium of rats].

OBJECTIVE: To compare the effects of ouabain and digoxin on both the systolic blood pressure (SBP) and sodium pump alpha-subunit expression in myocardium of rats. METHODS: Normal Sprague-Dawley (SD) rats were injected with ouabain, digoxin or normal saline (NS) everyday and indirect SBP was recorded once a week. Six weeks later, all the rats were killed and sodium pump alpha1-, alpha2-, and alpha3-subunit were detected in the left ventricular myocardium with RT-PCR method and immunohistochemical assay at mRNA and protein levels. RESULTS: At the end of six weeks SBP of rats infused with ouabain increased significantly (132.6 +/- 9.0 vs 115.7 +/- 8.2 mm Hg, P < 0.01), while no difference of SBP was found between digoxin group and NS group. The effects of ouabain and digoxin on sodium pump alpha-subunit isoform expression in myocardium were also different: both ouabain and digoxin stimulated expression of alpha3-isoform whereas alpha2-isoform unchanged at both mRNA and protein levels. alpha1-isoform was decreased in ouabain group and alpha1-isoform unchanged in digoxin group at both mRNA and protein levels. CONCLUSION: It is suggested that both ouabain and digoxin could regulate sodium pump alpha-subunit isoform expression, which might be related to the physiological roles of endogenous ouabain and might be responsible for the difference between the pharmacological and toxicological effects of ouabain and digoxin, including their effects on blood pressure.

Animals↗

PubMed2001

[Intact-bridge tympanomastoidectomy procedure: a reasonable option in the surgery of chronic otitis media and mastoiditis with cholesteatoma and/or granulation tissue].

OBJECTIVE: To study and evaluate a surgical option for the treatment of otitis media and mastoiditis with cholesteatoma and/or granulation tissue. METHODS: The procedure is called intact-bridge tympanomastoidectomy (IBM). There are some modifications for the treatment of patients suffered from chronic otitis media and mastoiditis with cholesteatoma and/or granulation tissue. The features of IBM include: 1. the aditus, mastoid and attic opened and the intractable pathological tissues eradicated thoroughly. 2. the posterior tympanal space opened through the facial recess if necessary. 3. a widened middle ear space by lowering bony bridge established. 4. the adults blocked. 5. immediate or staging tympanoplasties conducted according to the conditions of mucosa in the tympanum. RESULTS: The modified IBM procedure was performed on 62 ears from 61 cases. The follow-up has been 12-38 months and more than two years in 51 ears. The dry ear was obtained within 5-13 weeks with an average of 6 weeks. In 24.2% of ears the air-bone gap(ABG) was 20dB HL or less and within 21-30 dB HL in 41.9% of ears, the ABG over 31 dB HL was 16.1%. CONCLUSION: Our results indicate that IBM procedure fulfills the desired purposes of both open- and close-cavity techniques, the thorough eradication of diseased tissue allow to get a dry ear and restoration of hearing. IBM, therefore, is a reasonable choice for the surgery of otitis media and mastoiditis with cholesteatoma and/or granulation tissue.

Adolescent↗

PubMed2000

Antagonistic regulation of type I collagen gene expression by interferon-gamma and transforming growth factor-beta. Integration at the level of p300/CBP transcriptional coactivators.

Among the extracellular signals that modulate the synthesis of collagen, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) are preeminent. These two cytokines exert antagonistic effects on fibroblasts, and play important roles in the physiologic regulation of extracellular matrix turnover. We have shown previously that in normal skin fibroblasts, TGF-beta positively regulates alpha2(I) procollagen gene (COL1A2) promoter activity through the cellular Smad signal transduction pathway. In contrast, IFN-gamma activates Stat1alpha, down-regulates COL1A2 transcription, and abrogates its stimulation induced by TGF-beta. The level of integration of the two pathways mediating antagonistic collagen regulation is unknown. We now report that IFN-gamma abrogates TGF-beta-stimulated COL1A2 transcription in fibroblasts by inhibiting Smad activities. IFN-gamma appears to induce competition between activated Stat1alpha and Smad3 for interaction with limiting amounts of cellular p300/CBP. Overexpression of p300 restored COL1A2 stimulation by TGF-beta in the presence of IFN-gamma, and potentiated IFN-gamma-dependent positive transcriptional responses. In contrast to fibroblasts, in U4A cells lacking Jak1 and consequently unable to activate Stat1alpha-mediated responses, IFN-gamma failed to repress TGF-beta-induced transcription. These results indicate that as essential coactivators for both Smad3 and Stat1alpha, nuclear p300/CBP integrate signals that positively or negatively regulate COL1A2 transcription. The findings implicate a novel mechanism to account for antagonistic interaction of Smad and Jak-Stat pathways in regulation of target genes. In fibroblasts responding to cytokines with opposing effects on collagen transcription, the relative levels of cellular coactivators, and their interaction with regulated transcription factors, may govern the net effect.

Antineoplastic Agents↗

PubMed2000

Satellite and defective RNAs of Cryphonectria hypovirus 3-grand haven 2, a virus species in the family Hypoviridae with a single open reading frame.

Cryphonectria parasitica hypovirus 3-Grand Haven 2 (CHV3-GH2) is the most recently characterized member of the Hypoviridae family of viruses associated with hypovirulence of the chestnut blight fungus. Isolates of CHV3-GH2 contain either three or four double-stranded (ds) RNAs that are visible on ethidium bromide-stained agarose or polyacrylamide gels. Only the largest dsRNA appears to be required for virus infectivity, and was characterized previously (C. D. Smart et al., 1999, Virology 265, 66-73). In this study, we report the cloning, sequencing, and analysis of the other three dsRNAs. Sizes of the accessory dsRNAs are 3.6 kb (dsRNA 2), 1.9 kb (dsRNA 3), and 0.9 kb (dsRNA 4), compared to 9.8 kb for the genomic dsRNA segment (dsRNA 1). All three accessory dsRNA species are polyadenylated on the 3'-end of one strand, as is genomic dsRNA. DsRNA 2 represents a defective form of dsRNA 1, with the 5'-terminal 1.4 kb derived from the 5'-end of dsRNA 1 and the 3'-terminal 2.2 kb from the 3'-end of dsRNA 1. A single major open reading frame (ORF) is evident from deduced translations of dsRNA 2. The deduced translation product is a 91-kDa protein that represents a fusion consisting of the entire N-terminal protease and the entire putative helicase domain. DsRNAs 3 and 4 represent satellite RNAs that share very little sequence with dsRNA 1 and 2. DsRNA 4 is 937 nucleotides, excluding the poly(A)(+). The first AUG of the polyadenylated strand of dsRNA 4 occurs eight residues in from the 5'-terminus and would initiate the largest ORF on dsRNA 4, with the coding capacity for a 9.4-kDa protein. Within the deduced ORF and approximately 100 nucleotides from the 5'-end of dsRNA 4 is a 22-base sequence that is identical to sequences found in the nontranslated leaders of dsRNAs 1 and 2. DsRNA 3 accumulation in infected cultures varied, but it was less abundant than dsRNA 4. DsRNA 3 was found to represent a head-to-tail dimer of dsRNA 4 linked by a poly(A)/(U) stretch of 40-70 residues.

Amino Acid Sequence↗