Acute hantavirus infection induces galectin-3-binding protein.

Hantaviruses are zoonotic viruses that cause life-threatening diseases when transmitted to humans. Severe hantavirus infection is manifested by impairment of renal function, pulmonary oedema and capillary leakage. Both innate and adaptive immune responses contribute to the pathogenesis, but the underlying mechanisms are not fully understood. Here, we showed that galectin-3-binding protein (Gal-3BP) was upregulated as a result of hantavirus infection both in vitro and in vivo. Gal-3BP is a secreted glycoprotein found in human serum, and increased Gal-3BP levels have been reported in chronic viral infections and in several types of cancer. Our in vitro experiments showed that, whilst Vero E6 cells (an African green monkey kidney cell line) constitutively expressed and secreted Gal-3BP, this protein was detected in primary human cells only as a result of hantavirus infection. Analysis of Gal-3BP levels in serum samples of cynomolgus macaques infected experimentally with hantavirus indicated that hantavirus infection induced Gal-3BP also in vivo. Finally, analysis of plasma samples collected from patients hospitalized because of acute hantavirus infection showed higher Gal-3BP levels during the acute than the convalescent phase. Furthermore, the Gal-3BP levels in patients with haemorrhagic fever with renal syndrome correlated with increased complement activation and with clinical variables reflecting the severity of acute hantavirus infection.

Structural determinants of growth factor binding and specificity by VEGF receptor 2.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Hantavirus structure--molecular interactions behind the scene.

Viruses of the genus Hantavirus, carried and transmitted by rodents and insectivores, are the exception in the vector-borne virus family Bunyaviridae, since viruses of the other genera are transmitted via arthropods. The single-stranded, negative-sense, RNA genome of hantaviruses is trisegmented into small, medium and large (S, M and L) segments. The segments, respectively, encode three structural proteins: nucleocapsid (N) protein, two glycoproteins Gn and Gc and an RNA-dependent RNA-polymerase. The genome segments, encapsidated by the N protein to form ribonucleoproteins, are enclosed inside a lipid envelope that is decorated by spikes composed of Gn and Gc. The virion displays round or pleomorphic morphology with a diameter of roughly 120-160 nm depending on the detection method. This review focuses on the structural components of hantaviruses, their interactions, the mechanisms behind virion assembly and the interactions that maintain virion integrity. We attempt to summarize recent results on the virion structure and to suggest mechanisms on how the assembly is driven. We also compare hantaviruses to other bunyaviruses with known structure.

Inactivation of hantaviruses by N-ethylmaleimide preserves virion integrity.

Thiol groups of cysteine residues are crucial for the infectivity of various enveloped viruses, but their role in the infectivity of viruses of the family Bunyaviridae has thus far not been studied. This report shows that thiol groups are essential to the infectivity of hantaviruses. Alkylation of the thiol functional groups using the membrane-permeable compound N-ethylmaleimide (NEM) and membrane-impermeable compound 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) showed NEM to be a highly effective inactivator of Puumala and Tula hantaviruses. The NEM-inactivated hantavirus maintained the buoyant density of the wild-type virus. Furthermore, the antigenicity of glycoproteins and the cell attachment capacity of virions were retained at NEM concentrations that totally abolished virus infectivity. These results signified preservation of virion integrity following inactivation with NEM, making chemically inactivated virions valuable research antigens. It was demonstrated with biotin-conjugated maleimide, a mechanistic analogue of NEM, that all the structural proteins of hantavirus were sensitive towards thiol alkylation. In contrast to hantaviruses, NEM did not abolish Uukuniemi phlebovirus infectivity to the same extent. This indicates differences in the use of free thiols in virus entry among members of the family Bunyaviridae.

Interactions and oligomerization of hantavirus glycoproteins.

In this report the basis for the structural architecture of the envelope of hantaviruses, family Bunyaviridae, is systematically studied by the interactions of two glycoproteins N and C (Gn and Gc, respectively) and their respective disulfide bridge-mediated homo- and heteromeric oligomerizations. In virion extracts Gn and Gc associated in both homo- and hetero-oligomers which were, at least partially, thiol bridge mediated. Due to strong homo-oligomerization, the hetero-oligomers of Gn and Gc are likely to be mediated by homo-oligomeric subunits. A reversible pH-induced disappearance of a neutralizing epitope in Gc and dissociation of the Gn-Gc complex at pH values below 6.2 provide proteochemical evidence for the fusogenicity of Gc. Incomplete inactivation of virions at acidic pH indicates that additional factors are required for hantavirus fusion, as in the case of pestiviruses of the Flaviviridae. Based on similarities to class II fusion proteins, a structure model was created of hantavirus Gc using the Semliki Forest virus E1 protein as a template. In total, 10 binding regions for Gn were found by peptide scanning, of which five represent homotypic (Gn(I) to Gn(V)) and five represent heterotypic (Gc(I) to Gc(V)) interaction sites that we assign as intra- and interspike connections, respectively. In conclusion, the glycoprotein associations were compiled to a model wherein the surface of hantaviruses is formed of homotetrameric Gn complexes interconnected with Gc homodimers. This organization would create the grid-like surface pattern described earlier for hantaviruses in negatively stained electron microscopy specimens.

Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses.

Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, G(N) and G(C), are embedded in the viral membrane and form protrusions, or "spikes." The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the G(N)-G(C) spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus.

The cytoplasmic tails of envelope glycoprotein Gn of pathogenic hantaviruses but not of the apathogenic Prospect Hill virus (PHV) were recently reported to be proteasomally degraded in simian COS7 cells. Here, we show that the cytoplasmic tails of the glycoproteins of the apathogenic hantaviruses Tula virus (TULV) and PHV are also degraded through the ubiquitin-proteasome pathway, both in human HEK-293 and in simian Vero E6 cells. TULV Gn tails formed aggresomes in cells with proteasomal inhibitors. We conclude that degradation upon aggregation of Gn tails, which may represent a general cellular response to misfolded protein used by hantaviruses to control maturation of virions, is unrelated to pathogenicity.

Hantaviruses and TNF-alpha act synergistically to induce ERK1/2 inactivation in Vero E6 cells.

BACKGROUND: We have previously reported that the apathogenic Tula hantavirus induces apoptosis in Vero E6 epithelial cells. To assess the molecular mechanisms behind the induced apoptosis we studied the effects of hantavirus infection on cellular signaling pathways which promote cell survival. We previously also observed that the Tula virus-induced cell death process is augmented by external TNF-alpha. Since TNF-alpha is involved in the pathogenesis of hantavirus-caused hemorrhagic fever with renal syndrome (HFRS) we investigated its effects on HFRS-causing hantavirus-infected cells. RESULTS: We studied both apathogenic (Tula and Topografov) and pathogenic (Puumala and Seoul) hantaviruses for their ability to regulate cellular signaling pathways and observed a direct virus-mediated down-regulation of external signal-regulated kinases 1 and 2 (ERK1/2) survival pathway activity, which was dramatically enhanced by TNF-alpha. The fold of ERK1/2 inhibition correlated with viral replication efficiencies, which varied drastically between the hantaviruses studied. CONCLUSION: We demonstrate that in the presence of a cytokine TNF-alpha, which is increased in HFRS patients, hantaviruses are capable of inactivating proteins that promote cell survival (ERK1/2). These results imply that hantavirus-infected epithelial cell barrier functions might be compromised in diseased individuals and could at least partially explain the mechanisms of renal dysfunction and the resulting proteinuria seen in HFRS patients.

Human parvovirus B19 infection during pregnancy--value of modern molecular and serological diagnostics.

BACKGROUND: Over 95% of fetal complications (fetal hydrops and death) occur within 12 weeks following acute parvovirus B19 (B19) infection in pregnancy. Therefore, weekly fetal ultrasound monitoring is generally recommended for this time period. However, in the majority of women, typical symptoms of acute infection (rash or arthropathy) are absent, and during epidemics, B19 infection may be diagnosed incidentally by antibody screening of women at risk. OBJECTIVE: To assess the diagnostic value of currently available molecular and serological methods for reliable diagnosis of primary B19 infection in pregnancy. STUDY DESIGN: Large panels of well-characterized acute-phase or convalescent sera were used to investigate the ability of a VP2 IgM EIA, a Light-Cycler-based B19-DNA PCR, a VP1-IgG avidity EIA and two VP2-IgG epitope-type specificity [ETS] EIAs to pinpoint the time of primary B19 infection in pregnancy. RESULTS: The duration of low-level IgM positivity varied greatly (range 4-26 weeks). Samples collected within the first 2 weeks of infection showed high-level viremia (mean 1.75 x 10(8) geq/ml). During follow-up, low-level DNAemia (mean 9.7 x 10(4)geq/ml) persisted for at least 18 weeks in 91% (20/22) of patients. Considering the first 12 weeks after onset of disease the window of greatest risk for fetal complications, the "acute" phase was extended to cover this full period. In this case, performing the avidity and ETS-EIA sequentially, the positive predictive value was 100% in patients showing concordant avidity and ETS-EIA results. CONCLUSIONS: In the presence of low IgM titres and/or low-level DNAemia the use of supplementary serological assays such as VP1-IgG avidity EIA and VP2-ETS-EIA is advisable for restriction or avoidance of unnecessary fetal ultrasound examinations or invasive diagnostics; and in general for strengthening the reliability of B19 serodiagnosis of pregnant women.

Antibiotic properties of novel synthetic temporin A analogs and a cecropin A-temporin A hybrid peptide.

Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays. Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development. The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge.

Analysis of potato virus Y coat protein epitopes recognized by three commercial monoclonal antibodies.

BACKGROUND: Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs. CONCLUSIONS/SIGNIFICANCE: The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

The cytoplasmic tail of hantavirus Gn glycoprotein interacts with RNA.

We recently characterized the interaction between the intraviral domains of envelope glycoproteins (Gn and Gc) and ribonucleoprotein (RNP) of Puumala and Tula hantaviruses (genus Hantavirus, family Bunyaviridae). Herein we report a direct interaction between spike-forming glycoprotein and nucleic acid. We show that the envelope glycoprotein Gn of hantaviruses binds genomic RNA through its cytoplasmic tail (CT). The nucleic acid binding of Gn-CT is unspecific, as demonstrated by interactions with unrelated RNA and with single-stranded DNA. Peptide scan and protein deletions of Gn-CT mapped the nucleic acid binding to regions that overlap with the previously characterized N protein binding sites and demonstrated the carboxyl-terminal part of Gn-CT to be the most potent nucleic acid-binding site. We conclude that recognition of the RNP complex by the Gn-CT could be mediated by interactions with both genomic RNA and the N protein. This would provide the required selectivity for the genome packaging of hantaviruses.

Effective suppression of vascular network formation by combination of antibodies blocking VEGFR ligand binding and receptor dimerization.

Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy.

Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns.

Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary.

Impact of reductive cleavage of an intramolecular disulfide bond containing cationic gemini surfactant in monolayers and bilayers.

The properties of a novel disulfide-bond-containing gemini surfactant bis[N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide] disulfide (DSP) were studied using a Langmuir balance, supported monolayers, differential scanning calorimetry, giant vesicles, and LUVs. In 150 mM NaCl the cmc for DSP was 7.5 microM whereas that of the monomer N,N-dimethyl-N-hexadecyl-N-(2-mercaptoethyl)ammonium bromide (MSP) was 12.1 microM. Both surfactants exhibited single endotherms upon DSC, with peak temperatures Tm at 21.7 and 20.1 degrees C for DSP and MSP, respectively. The endotherm for MSP was significantly broader indicating less cooperative melting. Both in monolayers and in vesicles reductive cleavage of the disulfide bond of DSP could be obtained by glutathione (GSH). For Langmuir films of DSP the addition of GSH into the subphase led to a decrease in surface pressure pi as well as surface dipole potential psi. Although the cleavage by GSH was significantly slower in the presence of a charge saturating concentration of DNA, it did not prevent the reaction. The resulting monomers detached from supported monolayers, leading to loss of affinity of the surface for DNA. Disruption of giant vesicles containing DSP within approximately 30 s following a local injection of GSH was observed, revealing membrane destabilization.

Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells.

Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER-Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261-3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.

Apolipoprotein E activates the low-activity form of human phospholipid transfer protein.

Phospholipid transfer protein (PLTP) exists in a high-activity (HA-PLTP) and a low-activity form (LA-PLTP) in the circulation. LA-PLTP is associated with apoA-I while the HA-PLTP complex is enriched with apoE. To study the interaction of PLTP with apolipoproteins, we carried out surface plasmon resonance analyses. These demonstrated a concentration-dependent binding of recombinant human PLTP, which represents an active PLTP form, and LA-PLTP to apoE, apoA-I, and apoA-IV within a nanomolar K(D) range. To study whether LA-PLTP can be transformed into an active form, we incubated it in the presence of proteoliposomes containing apoE, apoA-I or apoA-IV. The apoE proteoliposomes induced a concentration-dependent activation of LA-PLTP. ApoA-IV proteoliposomes also activated LA-PLTP in a concentration-dependent manner, whereas apoA-I proteoliposomes had no such effect. These observations suggest that PLTP is capable of interacting with apoE, apoA-I, and apoA-IV, and that these interactions regulate PLTP-activity levels in plasma.

Expression of complement factor H binding immunoevasion proteins in Borrelia garinii isolated from patients with neuroborreliosis.

The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.

The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll.

The crystal structure of the recombinant collagen-binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 A resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel nine-coiled left-handed parallel beta-roll. Before the beta-roll, the polypeptide loops from one monomer to the rest, and after the beta-roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable 'lock nut' structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen-binding surface. Furthermore, a new structure-sequence motif for YadA beta-roll was used to identify putative YadA-head-like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.

Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation.

Hantaviruses are known to cause two severe human diseases: haemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. The mechanisms of pathogenesis of these two diseases are progressively becoming understood. Recently, two hantaviruses, Hantaan and Prospect Hill were reported to cause programmed cell death of Vero E6 cells. This study shows that Tula hantavirus (TULV) infection efficiently triggers an apoptotic programme in infected Vero E6 cells, and that the replication of TULV is required for the activation of caspase 3 and the cleavage of poly (ADP-ribose) polymerase, two molecular hallmarks of apoptosis. The enforced treatment of infected Vero E6 cells with tumour necrosis factor alpha (TNF-alpha), but not interferon alpha (IFN-alpha), advanced the time course of apoptosis. Furthermore, caspase 8 was activated on day 4 post-infection, the same day when caspase 3 was activated. TNF receptor 1 was induced during a late stage of TULV infection. These data suggest that, unlike during influenza A virus infection, TNF-alpha, but not type I IFN-alpha/beta, may contribute significantly to apoptosis in a synergistic manner with TULV propagation. Interestingly, pretreatment with a broad-spectrum caspase inhibitor, z-VAD-fmk, efficiently inhibited apoptosis of TULV-infected Vero E6 cells. Taken together, these results suggest that TULV replication initiates a typical apoptotic programme involving caspase 8 activation.