Seroprevalence of SARS-CoV-2 antibodies in the county town of Slovakia - a pilot study from the Trencin city.

Slovakia is a country with only 5.45 million inhabitants. However, the past two years of the COVID-19 pandemic have shown huge inter-regional differences. These were represented by different numbers of diagnosed SARS-CoV-2 cases and the vaccination rates in the regions, as well as by the willingness of the inhabitants to comply with anti-pandemic measures or to undergo testing. The occurrence of such regional disparities provided a rational basis for monitoring the epidemic situation within smaller areas, e.g. at city level. Trencin is a medium-sized Slovak county town with about 55 000 inhabitants. The city administration gave its residents the opportunity to assess their current level of antibodies against the SARS-CoV-2 virus, and received an additional benefit in the form of data on the real epidemic situation in the city, which helped in further management of anti-pandemic measures. The primary aim of the study, conducted in January and February 2022, was to determine the levels of antibodies against the SARS-CoV-2 virus in the inhabitants of Trencin. The results showed that 75% of the study participants, representing the adult population of the city, had detectable IgG antibodies against the SARS-CoV-2 spike protein. Noteworthy, at the time of the study, 13% of the Trencin city population who were unaware of overcoming COVID-19 had specific antibodies against the virus. Furthermore, the antibody levels in recovered unvaccinated subjects increased not only with the severity of their COVID-19 symptoms, but also after multiple recoveries from the disease. On the other hand, the severity of side effects after vaccination did not influence the antibody levels. The results of the study are in line with the current view that hybrid immunity (vaccination plus SARS-CoV-2 infection in any order) offers greater protection than immunity elicited by vaccination or COVID-19 separately. Keywords: SARS-CoV-2 coronavirus; COVID-19; ELISA; seroprevalence; antibodies; vaccination.

Seroprevalence of SARS-CoV-2 IgG antibodies in the staff of the Slovak Academy of Sciences in response to COVID-19 and/or vaccination: situation in August 2021.

Cross-sectional seroprevalence study of SARS-CoV-2 IgG antibodies was accomplished in the Slovak Academy of Sciences to inform authorities of research institutions about the situation at their workplaces, to assess the risk of next exposure to SARS-CoV-2, and to guide decisions on institutional measures sustaining essential research in evolving epidemic situation. Study participants provided informed consent, anamnestic information, and self-collected dry blood spot samples that were analyzed by ELISA for SARS-CoV-2 S protein-specific IgG antibodies. Relative antibody levels detected in 1928 subjects showed seroprevalence of 84.13% and led to the following main findings consistent with the current knowledge: (1) mRNA-based vaccines induce better humoral response compared to adenovirus vaccines, (2) antibody levels reflect severity of COVID-19 symptoms, (3) post-COVID vaccination results in marked elevation of IgG levels particularly in asymptomatic and mild cases, (4) antibody levels decrease with increasing time elapsed from vaccination or COVID-19. In addition, data sorting to distinct research institutes and their clustering to three principal scientific sections of the Slovak Academy of Sciences revealed marked differences in seroprevalence, and allowed to identify workplaces with relatively high seropositivity and response rate that can potentially provide a safer working environment than those, where seroprevalence was low or unknown due to low participation. Thus, findings of this study can have direct implications on management decisions during the next pandemic development, with the necessity to keep in mind the phenomenon of time-dependent immunity waning and current spread of more contagious Delta variant of SARS-CoV-2. Keywords: SARS-CoV-2 coronavirus; COVID-19; spike protein; seroprevalence; antibodies; vaccination.

Carbonic Anhydrase IX-Mouse versus Human.

In contrast to human carbonic anhydrase IX (hCA IX) that has been extensively studied with respect to its molecular and functional properties as well as regulation and expression, the mouse ortholog has been investigated primarily in relation to tissue distribution and characterization of CA IX-deficient mice. Thus, no data describing transcriptional regulation and functional properties of the mouse CA IX (mCA IX) have been published so far, despite its evident potential as a biomarker/target in pre-clinical animal models of tumor hypoxia. Here, we investigated for the first time, the transcriptional regulation of the Car9 gene with a detailed description of its promoter. Moreover, we performed a functional analysis of the mCA IX protein focused on pH regulation, cell-cell adhesion, and migration. Finally, we revealed an absence of a soluble extracellular form of mCA IX and provided the first experimental evidence of mCA IX presence in exosomes. In conclusion, though the protein characteristics of hCA IX and mCA IX are highly similar, and the transcription of both genes is predominantly governed by hypoxia, some attributes of transcriptional regulation are specific for either human or mouse and as such, could result in different tissue expression and data interpretation.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Encapsulation of anti-carbonic anhydrase IX antibody in hydrogel microspheres for tumor targeting.

Encapsulation is a well-established method of biomaterial protection, controlled release, and efficient delivery. Here we evaluated encapsulation of monoclonal antibody M75 directed to tumor biomarker carbonic anhydrase IX (CA IX) into alginate microbeads (SA-beads) or microcapsules made of sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) (PMCG). M75 antibody release was quantified using ELISA and its binding properties were assessed by immunodetection methods. SA-beads showed rapid M75 antibody release in the first hour, followed by steady release during the whole experiment of 7 days. In contrast, the M75 release from PMCG capsules was gradual, reaching the maximum concentration on the 7th day. The release was more efficient at pH 6.8 compared to pH 7.4. The released antibody could recognize CA IX, and target the CA IX-positive cells in 3D spheroids. In conclusion, SA-beads and PMCG microcapsules can be considered as promising antibody reservoirs for targeting of cancer cells.

Expression pattern of carbonic anhydrase IX in Medullary thyroid carcinoma supports a role for RET-mediated activation of the HIF pathway.

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.

Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain.

Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.

Hypoxia induces the gene expression and extracellular transmission of persistent lymphocytic choriomeningitis virus.

The physiological context of virus-infected cells can markedly affect multiplication and spread of the virus progeny. During persistent infection, the virus exploits the host cell without disturbing its vital functions. However, microenvironmental hypoxia can uncouple this intimate relationship and escalate virus pathogenesis. Accumulating evidence suggests that hypoxia-inducible factor (HIF) modulates gene expression of the viruses that pass through a DNA stage, contain hypoxia-responsive promoter elements, and replicate in the nucleus. Here we show that hypoxia can influence the gene expression and transmission of the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV), which is a neglected human pathogen and teratogen. The MX strain of LCMV, which we used as a model, replicates in a persistent mode in human HeLa cells, fails to produce mature envelope glycoproteins, and spreads through cell-cell contacts in the absence of extracellular infectious virions. Both exposure of MX-infected HeLa cells to chronic hypoxia and gene transfer approaches led to increased virus RNA transcription and higher levels of the viral proteins via a HIF-dependent mechanism. Moreover, hypoxia enhanced the formation of infectious virions capable of transmitting LCMV by cell-free medium. This LCMV "reactivation" might have health-compromising consequences in hypoxia-associated situations, such as fetal development and ischemia-related pathologies.

Ferrate (VI), Fenton Reaction and Its Modification: An Effective Method of Removing SARS-CoV-2 RNA from Hospital Wastewater.

The outbreak of the coronavirus disease 2019 (COVID-19) raises questions about the effective inactivation of its causative agent, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in medical wastewater by disinfectants. For this reason, our study of wastewater from a selected hospital evaluated several different advanced oxidation methods (Fenton reaction and Fenton-like reaction and ferrate (VI)) capable of effectively removing SARS-CoV-2 RNA. The obtained results of all investigated oxidation processes, such as ferrates, Fenton reaction and its modifications achieved above 90% efficiency in degradation of SARS-CoV-2 RNA in model water. The efficiency of degradation of real SARS-CoV-2 from hospital wastewater declines in following order ferrate (VI) > Fenton reaction > Fenton-like reaction. Similarly, the decrease of chemical oxygen demand compared to effluent was observed. Therefore, all of these methods can be used as a replacement of chlorination at the wastewater effluent, which appeared to be insufficient in SARS-CoV-2 removal (60%), whereas using of ferrates showed efficiency of up to 99%.

Apoptosis induced clustering of IP(3)R1 in nuclei of non-differentiated PC12 cells.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.

Glucocorticoid receptor-mediated transcriptional activation of S100P gene coding for cancer-related calcium-binding protein.

S100P is a member of the S100 family of calcium-binding proteins involved in calcium sensing and signal transduction. Its abnormal expression and biological activities are linked to tumor phenotype, namely to increased survival, proliferation, invasion and metastatic propensity of tumor cells. Association of S100P with outcome of tumor treatment and preliminary data from S100P promoter analysis prompted us to study regulation of S100P expression by glucocorticoids, which are implicated in tumor response to chemotherapy. We showed that dexamethasone (DX), a representative glucocorticoid, was capable to induce activity of S100P promoter by means of increased expression, nuclear translocation, and transactivation properties of the glucocorticoid receptor (GR). Moreover, DX treatment led to decreased phosphorylation of ERK1/2, reduced transcriptional activity of AP1, and modulated activity of some additional transcription factors. We identified a promoter region responsible for DX-mediated transactivation and proved GR binding to S100P promoter. We found that the effect of DX was enhanced by partial but not complete inhibition of the MAPK/ERK pathway, supporting an active crosstalk between GR and MAPK/ERK signal transduction in control of S100P expression. On the other hand, suppression of GR mRNA level by transient siRNA expression resulted in reduced S100P transcription. The role of GR activation in S100P regulation was supported by co-expression of GR with S100P in cells treated with DX. These data suggest that S100P is a direct transcriptional target of glucocorticoid-mediated signaling in tumor cells that is activated through the interplay of GR and MAPK pathways.

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α.

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.

Sorption of SARS-CoV-2 Virus Particles to the Surface of Microplastics Released during Washing Processes.

The research aims at washing processes as possible sources of microplastics, specifical microfibers in wastewater, and the behavior of the virus particles SARS-CoV-2 in wastewater after the washing process as well as their ability to sorb to the surface of microfibers, released from washing processes. The conclusions of the research point to the ability of the virus to attach to possible solid impurities such as textile fibers (microfibers) occurring in the sewer and to the ability of wash water to influence their possible occurrence in the sewer. The highest efficiency (more than 99%) of removal virus particles was after washing process, using liquid washing powder, and washing soda. These findings may gradually contribute to a better understanding of the behavior of the virus particles in the sewer.

The effect of surface modification of TiMg composite on the in-vitro degradation response, cell survival, adhesion, and proliferation.

This study is aimed to evaluate the influence of mechanical surface treatment on the degradation response, cell survival, adhesion, and proliferation of a TiMg composite material. Two sets of the TiMg samples with different surface characteristics were studied: i) as-machined samples (TiMg-T) and ii) samples with a mechanically modified surface (TiMg-P). Surface roughness was determined using a confocal microscope. Degradation rates (DR) were evaluated in artificial Plasma, HBSS, and NaCl 0.9%. The cell viability was evaluated using an MTT assay. The initial cell adhesion and spreading were investigated using the direct contact assay. An xCELLigence system was employed to provide real-time cell proliferation. The focal adhesion and cell morphological changes were also examined. The DR of TiMg-P decreased by ⁓5 times compared with that of TiMg-T. Surface of the TiMg-P specimens after 72 h exposure to either HBSS or Plasma was passivated by a layer enriched with bioactive Ca/P species. The cell viability of L929 and Saos-2 after 72 h incubation for TiMg-P was 94.6% and 94.8% compared with 73.8% and 74.3% obtained for TiMg-T, respectively. The direct contact assay showed that the initial adhesion and spreading of the L929 cells incubated with TiMg-P was more pronounced compared with that of TiMg-T. The proliferation rate of Saos-2 cells incubated with TiMg-P was higher when compared with that of TiMg-T, and was almost comparable to that of the DMEM-blank between the 24 and 72 h interval. TiMg-P had a pronounced difference in the number and area of Focal Adhesions (FA) compared with that of TiMg-T. The morphology of cells incubated with TiMg-P was not altered. The results confirmed that the smooth and less strained surface of the TiMg-P samples effectively improved the in-vitro degradation response, cell survival, adhesion, and proliferation.

Role of the HBx oncoprotein in carbonic anhydrase 9 induction.

Carbonic anhydrase 9 (CA9), as one of the most hypoxia-responsive genes, has been associated almost exclusively with hypoxic tumors. Its principal role is in pH regulation which helps tumor cells overcome intracellular acidosis and survive extended periods of time with low oxygen. Hypoxia-inducible factor 1 (HIF-1) is the main transcriptional activator of CA9. Hepatitis B virus X protein (HBx) has been shown to increase the transcriptional activity of HIF-1. HBx is often expressed from the gene integrated in the hepatocytes infected persistently and contributes significantly to alterations in host gene expression that can lead to the development of hepatocellular carcinoma (HCC) associated with Hepatitis B virus (HBV). The aim of this study was to determine the effect of HBx on expression of CA9. Transient transfection of HBx led to an increase in the expression of CA9 as assessed by RT-PCR and Western blotting. HBx was able to increase CA9 promoter activity significantly in several cell lines. The effect was mediated via HIF-1 and a functional HRE element located -10/-3 bp upstream of the CA9 transcription initiation site. These data suggest that CA9 may be involved in the development of HCC by contributing to the survival of hepatocytes infected with HBV in liver tissue with fibrosis.

Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX.

Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1alpha and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1alpha competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.

Clinical and Pre-Clinical Evidence of Carbonic Anhydrase IX in Pancreatic Cancer and Its High Expression in Pre-Cancerous Lesions.

Hypoxia is a common phenomenon that occurs in most solid tumors. Regardless of tumor origin, the evolution of a hypoxia-adapted phenotype is critical for invasive cancer development. Pancreatic ductal adenocarcinoma is also characterized by hypoxia, desmoplasia, and the presence of necrosis, predicting poor outcome. Carbonic anhydrase IX (CAIX) is one of the most strict hypoxia regulated genes which plays a key role in the adaptation of cancer cells to hypoxia and acidosis. Here, we summarize clinical data showing that CAIX expression is associated with tumor necrosis, vascularization, expression of Frizzled-1, mucins, or proteins involved in glycolysis, and inevitably, poor prognosis of pancreatic cancer patients. We also describe the transcriptional regulation of CAIX in relation to signaling pathways activated in pancreatic cancers. A large part deals with the preclinical evidence supporting the relevance of CAIX in processes leading to the aggressive behavior of pancreatic tumors. Furthermore, we focus on CAIX occurrence in pre-cancerous lesions, and for the first time, we describe CAIX expression within intraductal papillary mucinous neoplasia. Our review concludes with a detailed account of clinical trials implicating that treatment consisting of conventionally used therapies combined with CAIX targeting could result in an improved anti-cancer response in pancreatic cancer patients.

CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia.

Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO2 to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cells in vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD+ and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO2 to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.

Up-regulation of Murid herpesvirus 4 ORF50 by hypoxia: possible implication for virus reactivation from latency.

Murid herpesvirus 4 (MuHV-4) is a member of the Gammaherpesvirus subfamily capable to establish a long-lasting latency and induce occasional malignancies. Because MuHV-4 is associated with cancer in a subset of virus-infected mice and because tumor development is often linked with hypoxia, we studied the influence of hypoxia on the biology of this virus. Using immunofluorescence and FACS analysis we detected increased proportion of MuHV-4 positive cells in the latently infected NB-78 cell line exposed to low oxygen conditions compared to normoxic controls. Moreover, the expression of ORF50, a crucial gene responsible for switch from latency to lytic virus replication, was induced upon the exposure of NB-78 cells to hypoxia. Luciferase reporter assays with ORF50 promoter confirmed the hypoxia-dependent induction. Transient co-transfections with hypoxia inducible factors showed that HIF-2alpha is a more potent activator of ORF50 expression than HIF-1alpha. Our results confirm that the MuHV-4 life cycle is influenced by low oxygen concentration.

Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus.

The N-terminal region of a 32 kDa cell-surface-binding protein, encoded by the D8L gene of vaccinia virus, shows sequence homology to CAs (carbonic anhydrases; EC 4.2.1.1). The active CAs catalyse the reversible hydration of CO2 to bicarbonate participating in many physiological processes. The CA-like domain of vaccinia protein [vaccCA (vaccinia virus CA-like protein)] contains one of the three conserved histidine residues required for co-ordination to the catalytic zinc ion and for enzyme activity. In the present study, we report the engineering of catalytically active vaccCA mutants by introduction of the missing histidine residues into the wild-type protein. The wild-type vaccCA was inactive as a catalyst and does not bind sulfonamide CA inhibitors. Its position on a phylogram with other hCAs (human CAs) shows a relationship with the acatalytic isoforms CA X and XI, suggesting that the corresponding viral gene was acquired from the human genome by horizontal gene transfer. The single mutants (vaccCA N92H/Y69H) showed low enzyme activity and low affinity for acetazolamide, a classical sulfonamide CA inhibitor. The activity of the double mutant, vaccCA N92H/Y69H, was much higher, of the same order of magnitude as that of some human isoforms, namely CA VA and CA XII. Moreover, its affinity for acetazolamide was high, comparable with that of the most efficient human isoenzyme, CA II (in the low nanomolar range). Multiplication of vaccinia virus in HeLa cells transfected with the vaccCA N92H/Y69H double mutant was approx. 2-fold more efficient than in wild-type vaccCA transfectants, suggesting that the reconstitution of the enzyme activity improved the virus life cycle.