Replication and gene function in Kunjin virus.
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Animals↗
Biomedical subjects
J M Mackenzie
Publications and source records attributed to J M Mackenzie.
Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection.
Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles, but at times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.
Animals↗
Comparison of breastfeeding rates in Scotland in 1990-1 and 1997-8.
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Adult↗
Stable expression of noncytopathic Kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of Kunjin virus.
This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GalT. Importantly, persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in virus infections.
Animals↗
Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively.
The intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (RER). Building on previous studies involving immunofluorescence (IF) and cryoimmunoelectron microscopy of Kunjin virus (KUN)-infected cells, we sought to identify the steps involved in the assembly and maturation of KUN. Thus, using antibodies directed against envelope protein E in IF analysis, we found the accumulation of E within regions coincident with the RER and endosomal compartments. Immunogold labeling of cryosections of infected cells indicated that E and minor envelope protein prM were localized to reticulum membranes continuous with KUN-induced convoluted membranes (CM) or paracrystalline arrays (PC) and that sometimes the RER contained immunogold-labeled virus particles. Both proteins were also observed to be labeled in membranes at the periphery of the induced CM or PC structures, but the latter were very seldom labeled internally. Utilizing drugs that inhibit protein and/or membrane traffic throughout the cell, we found that the secretion of KUN particles late in infection was significantly affected in the presence of brefeldin A and that the infectivity of secreted particles was severely affected in the presence of monensin and N-nonyl-deoxynojirimycin. Nocodazole did not appear to affect maturation, suggesting that microtubules play no role in assembly or maturation processes. Subsequently, we showed that the exit of intact virions from the RER involves the transport of individual virions within individual vesicles en route to the Golgi apparatus. The results suggest that the assembly of virions occurs within the lumen of the RER and that subsequent maturation occurs via the secretory pathway.
Animals↗
Intrascleral recurrence of uveal melanoma after transretinal "endoresection".
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Adult↗
Breastfeeding rates are increasing in Scotland.
OBJECTIVE: To measure the change in prevalence of breastfeeding between 1990/1991 and 1997/1998 in Scotland, using information collected on Guthrie cards when newborn infants are about seven days old. DESIGN: Analysis, by geographic postcode area, health board and maternity unit, for babies born in 1990/1991 and 1997/1998. For 1997, maternity unit and health board breastfeeding rates were also compared after standardisation for maternal age, deprivation and age of infant. SETTING: Scotland. SUBJECTS: 131,759 babies born in 1990/1991 and 118,055 in 1997/1998. RESULTS: In 1990/1991, 46,949 (35.6%) were breastfed as were 49,615 (42.0%) in 1997/1998, an increase of 6.4% (95% CI 6.0, 6.8) over eight years. A 3.8% increase remained after adjustment for change in maternal age. Maternity units with the Baby Friendly award improved 8.1% (95% CI 7.0, 9.2) compared with those with a certificate of commitment 6.1% (95% CI 5.2, 7.0). Other units improved 2.2% (95% C1 1.6, 2.8) no more than estimates due to increase in maternal age. Standardised rates were higher on the East Coast of Scotland 111 (109, 112) than the West or Central Regions 97 (96, 99). CONCLUSION: Breastfeeding has increased over eight years in Scotland. Less than half can be explained by demographic change in maternal age. However present breastfeeding targets are unlikely to be met. Maternity units should be urged to participate fully in the UNICEF U.K. Baby Friendly Initiative. Effective interventions prior to pregnancy are required so that more young men and women want their babies to be breastfed.
Breast Feeding↗
Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.
Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.
Amino Acid Sequence↗
School based screening for hypothyroidism in Down's syndrome by dried blood spot TSH measurement.
OBJECTIVE: To determine the feasibility of annual hypothyroid screening of children with Down's syndrome by measuring thyroid stimulating hormone (TSH) on dried blood spots at school, and to describe the outcome in positive children. DESIGN: Establishment of a register of school children with Down's syndrome, and procedures for obtaining permission from parents, annual capillary blood samples, TSH measurement, and clinical assessment of children with TSH values > 10 mU/litre. SUBJECTS: All school age children with Down's syndrome within Lanarkshire and Glasgow Health Boards during 1996-7 and 1997-8. RESULTS: 200 of 214 school children with Down's syndrome were screened. Four of the unscreened children were receiving thyroxine treatment, and only 5 remained unscreened by default. 15 of the 200 children had capillary TSH > 10 mU/litre, and all but 1 had evidence of Hashimoto's thyroiditis. Seven of the 15 children started thyroxine treatment immediately, 6 with a pronounced rise in venous TSH and subnormal free thyroxine (fT4), and one with mildly raised TSH and normal fT4 but symptoms suggesting hypothyroidism. Eight children with mildly raised venous TSH and normal fT4 were left untreated; 1 year after testing positive, fT4 remained > 9 pmol/litre in all cases, but 4 children were started on thyroxine because of a rise in TSH. TSH fell in 3 of the 4 remaining children and there was a marginal rise in 1; all remain untreated. The prevalence of thyroid disease in this population is >/= 8.9%. CONCLUSION: Dried blood spot TSH measurement is effective for detecting hypothyroidism in Down's syndrome and capillary sampling is easily performed at school. The existing programme could be extended to the whole of Scotland within a few years.
Adolescent↗
Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1.
The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice.
Amino Acid Sequence↗
Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes.
Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitored in situ by immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G., Virology 140, 68-79, 1985). The model assumes that there is on average only one nascent strand per template, which remains bound until displaced during the next cycle of RNA synthesis. The replicase complex located by BrU incorporation in the identified foci was stable, remaining active in incorporating BrU or [32P]orthophosphate in viral RNA after complete inhibition of protein synthesis in cycloheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immunogold labeling of induced vesicle packets is functioning as the true replicative intermediate (Westaway et al., J. Virol. 71, 6650-6661, 1997; Mackenzie et al., Virology 245, 203-215, 1998). Implications are that the replicase complex is able to recycle in the same membrane site in the absence of continuing protein synthesis and that possibly apart from uncleaved NS3-NS4A, it has no requirement for a polyprotein precursor late in infection.
Animals↗
Markers for trans-Golgi membranes and the intermediate compartment localize to induced membranes with distinct replication functions in flavivirus-infected cells.
Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.
Animals↗
Subcellular localization and some biochemical properties of the flavivirus Kunjin nonstructural proteins NS2A and NS4A.
In a previous study on the replication of Kunjin virus using immunoelectron microscopy (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, 1997, J. Virol. 71, 6650-6661), NS1 and NS3 were found associated with double-stranded RNA (dsRNA) within vesicle packets (VP) in infected Vero cells, suggesting that these induced membrane structures may be the cytoplasmic sites of RNA replication. NS2B and NS3 (comprising the virus-encoded protease) were colocalized within distinct paracrystalline (PC) or convoluted membranes (CM), also induced in the cytoplasm, suggesting that these membranes are the sites of proteolytic cleavage. In this study we found by immunofluorescence (IF) that the small hydrophobic nonstructural proteins NS2A and NS4A were located in discrete foci in the cytoplasm of infected cells at both 16 and 24 h postinfection, partially coincident with dsRNA foci. In cryosections of infected cells at 24 h, NS2A was located by immunogold labeling primarily within VP, associated with labeled dsRNA. NS2A fused to glutathione S-transferase (GST) bound strongly to the 3' untranslated region of Kunjin RNA and also to the proposed replicase components NS3 and NS5 in cell lysates. NS4A was localized by immunogold labeling within a majority of the virus-induced membranes, including VP, CM, and PC. GST-NS4A bound weakly to the 3' untranslated region of Kunjin RNA but was bound to NS4A strongly and to most of the other viral nonstructural proteins, including NS3 and NS5. Taken together the results indicate that the flavivirus replication complex includes NS2A and NS4A in the VP in addition to the previously identified NS1 and NS3.
Animals↗
New variant Creutzfeldt-Jakob disease: neurological features and diagnostic tests.
BACKGROUND: In April, 1996, ten cases of Creutzfeldt-Jakob disease (CJD) with an apparently new clinicopathological phenotype were published and it was suggested that these new variant cases (nvCJD) might be causally linked to bovine spongiform encephalopathy (BSE). There have now been 21 cases of nvCJD in the UK and one case in France. We report clinical features and diagnostic test results of the first 14 cases of nvCJD in the UK. METHODS: Case ascertainment of CJD was mainly by direct referral from neurologists and neuropathologists. Clinical and investigate details were obtained by interview with patients' relatives and by examination of case notes. Ten cases in this report were examined while alive. Prion protein (PrP) gene analysis was carried out with informed consent from the patient or from a relative. The diagnosis of nvCJD was established histologically. FINDINGS: Eight cases were women. Mean age at onset of symptoms was 29 (16-48) years and the median duration of illness was 14 (9-35) months. All patients had early psychiatric symptoms, most often depression, and 13 were seen by a psychiatrist early in the clinical course. Eight patients developed early sensory symptoms which were persistent and often painful. Neurological signs, including ataxia and involuntary movements, developed in all cases and towards the end of the illness, most had akinetic mutism. The electroencephalogram was abnormal in most patients but typical periodic complexes of CJD were not seen in any case. Cerebral imaging was usually normal or showed non-specific abnormalities; in two cases magnetic-resonance imaging scans showed high signal in the thalamus. INTERPRETATION: Clinical features in these cases are similar and relatively distinct from other forms of CJD, suggesting that this is a new clinical phenotype consistent with a single strain of infectious agent. There is, however, some overlap with atypical cases of sporadic CJD, and the diagnosis of nvCJD remains dependent on neuropathological confirmation.
Adolescent↗
Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus.
The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.
Animals↗
Case report: inflammatory pseudotumour of the spine, with literature review.
A case of inflammatory pseudotumour of the thoracic spine is reported. There appears to be only one previous report of a spinal case. The patient, a 58-year-old woman, presented with symptoms and signs of spinal cord compression indistinguishable from those due to extradural tumour. The radiological, operative and pathological findings are presented together with a literature review.
Decompression, Surgical↗
Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.
The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.
Animals↗
Bleb formation and F-actin distribution during mitosis and tumor necrosis factor-induced apoptosis.
The murine cell line C3HA has been used extensively in studies of the cytopathology that accompanies TNF-induced cytolysis. This cell line undergoes an apoptic form of cell death characterized by plasma membrane blebbing and cytoplasmic boiling. Since plasma membrane blebs also appear on C3HA cells during mitosis, in this report we have compared these blebs with those that appear during apoptosis to determine whether they represent related structures. Our results reveal several differences. During mitosis, the blebs that appear are smaller and more heterogeneous in size than are those that appear during apoptosis. In addition, during mitosis bleb formation is preceded by the appearance of microvilli on the cell surface. No microvilli are observed during apoptosis. The staining pattern with rhodamine phalloidin also differed between mitotic and apoptic blebs, indicating a difference in their content of f-actin. The blebs that form during mitosis stained in a bright, uniform manner, suggesting an association with f-actin. In contrast, apoptic blebs were stained only at their base, the bleb itself being devoid of f-actin-associated staining. This difference may help explain why mitotic blebs are reintegrated into the cell surface, while the blebs that form during apoptosis are not.
Actins↗