Insight into the Hantaan virus RNA-dependent RNA polymerase inhibition using in-silico approaches.

The Hantaan virus (HTN) is a member of the hantaviridae family. It is a segmented type, negative-strand virus (sNSVs). It causes hemorrhagic fever with renal syndrome, which includes fever, vascular hemorrhage, and renal failure. This illness is one of the most serious hemorrhagic diseases in the world, and it is a major public health concern due to its high mortality rate. The Hantaan virus RNA-dependent RNA polymerase complex (RdRp) is involved in viral RNA transcription and replication for the survival and transmission of this virus. Therefore, it is a primary target for antiviral drug development. Interference with the endonucleolytic "cap-snatching" reaction by the HTN virus RdRp endonuclease domain is a particularly appealing approach for drug discovery against this virus. This RdRp endonuclease domain of the HTN virus has a metal-dependent catalytic activity. We targeted this metal-dependent enzymatic activity to identify inhibitors that can bind and disrupt this endonuclease enzyme activity using in-silico approaches i.e., molecular docking, molecular dynamics simulation, predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) and drug-likeness studies. The docking studies showed that peramivir, and ingavirin compounds can effectively bind with the manganese ions and engage with other active site residues of this protein. Molecular simulations also showed stable binding of these ligands with the active site of HTN RdRp. Simulation analysis showed that they were in constant contact with the active site manganese ions and amino acid residues of the HTN virus endonuclease domain. This study will help in better understanding the HTN and related viruses.

Hantaan virus inhibits type I interferon response by targeting RLR signaling pathways through TRIM25.

Hantaan virus (HTNV) is responsible for hemorrhagic fever with renal syndrome (HFRS), primarily due to its ability to inhibit host innate immune responses, such as type I interferon (IFN-I). In this study, we conducted a transcriptome analysis to identify host factors regulated by HTNV nucleocapsid protein (NP) and glycoprotein. Our findings demonstrate that NP and Gc proteins inhibit host IFN-I production by manipulating the retinoic acid-induced gene I (RIG-I)-like receptor (RLR) pathways. Further analysis reveals that HTNV NP and Gc proteins target upstream molecules of MAVS, such as RIG-I and MDA-5, with Gc exhibiting stronger inhibition of IFN-I responses than NP. Mechanistically, NP and Gc proteins interact with tripartite motif protein 25 (TRIM25) to competitively inhibit its interaction with RIG-I/MDA5, suppressing RLR signaling pathways. Our study unveils a cross-talk between HTNV NP/Gc proteins and host immune response, providing valuable insights into the pathogenic mechanism of HTNV.

Hantaan virus nucleocapsid protein stimulates MDM2-dependent p53 degradation.

Apoptosis has been shown to be induced and downregulated by the Hantaan virus (HTNV) nucleocapsid (N) protein. To address these conflicting data, expression of the p53 protein, one of the key molecules involved in apoptosis, was assessed in the presence of the N protein in A549 and HeLa cells. The amount of p53, increased by drug treatment, was reduced when cells were infected with HTNV or transfected with an expression vector of the HTNV N protein. When cells were treated with a proteasome inhibitor (MG132) or an MDM2 antagonist (Nutlin-3), p53 expression was not reduced in N protein-overexpressed cells. We concluded that the HTNV N protein ubiquitinates and degrades p53 MDM2-dependently. Here we report downregulation of p53 expression through a post-translational mechanism: MDM2-dependent ubiquitination and degradation by the HTNV N protein. These results indicate that N protein-dependent p53 degradation through the ubiquitin proteasome system is one of the anti-apoptotic mechanisms employed by HTNV.

STING strengthens host anti-hantaviral immunity through an interferon-independent pathway.

Hantaan virus (HTNV), the prototype virus of hantavirus, could escape innate immunity by restraining type I interferon (IFN) responses. It is largely unknown whether there existed other efficient anti-hantaviral tactics in host cells. Here, we demonstrate that the stimulator of interferon genes (STING) strengthens the host IFN-independent anti-hantaviral immunity. HTNV infection activates RIG-I through IRE1-XBP 1-mediated ER stress, which further facilitates the subcellular translocation and activation of STING. During this process, STING triggers cellular autophagy by interacting with Rab7A, thus restricting viral replication. To note, the anti-hantaviral effects of STING are independent of canonical IFN signaling. Additionally, neither application of the pharmacological antagonist nor the agonist targeting STING could improve the outcomes of nude mice post HTNV challenge in vivo. However, the administration of plasmids exogenously expressing the mutant C-terminal tail (ΔCTT) STING, which would not trigger the type I IFN responses, protected the nude mice from lethal HTNV infection. In summary, our research revealed a novel antiviral pathway through the RIG-I-STING-autophagy pathway, which offered novel therapeutic strategies against hantavirus infection.

Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5' termini of their genomic RNAs are monophosphorylated.

dsRNA and 5'-triphosphate RNA are considered critical activators of the innate immune response because of their interaction with pattern recognition receptors. It has been reported that no dsRNA is detected in negative-sense RNA virus-infected cells and that Hantaan virus (HTNV) genomic RNA bears a 5' monophosphate group. In this paper we examine the 5' termini of genomic RNAs of and dsRNA production by two major groups of Old World hantaviruses. No detectable amounts of dsRNA were found in infected cells. Also, the genomic RNAs of these hantaviruses bear a 5' monophosphate group and therefore are unable to trigger interferon induction. Taken together with the earlier data on HTNV, these results suggest that in addition to the dsRNA and genomic RNA, which may be only minimally involved in the induction of innate immunity, other cellular signalling pathways may also be involved and that these await further investigation.

Pathogenic hantaviruses Andes virus and Hantaan virus induce adherens junction disassembly by directing vascular endothelial cadherin internalization in human endothelial cells.

Hantaviruses infect endothelial cells and cause 2 vascular permeability-based diseases. Pathogenic hantaviruses enhance the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF). However, the mechanism by which hantaviruses hyperpermeabilize endothelial cells has not been defined. The paracellular permeability of endothelial cells is uniquely determined by the homophilic assembly of vascular endothelial cadherin (VE-cadherin) within adherens junctions, which is regulated by VEGF receptor-2 (VEGFR2) responses. Here, we investigated VEGFR2 phosphorylation and the internalization of VE-cadherin within endothelial cells infected by pathogenic Andes virus (ANDV) and Hantaan virus (HTNV) and nonpathogenic Tula virus (TULV) hantaviruses. We found that VEGF addition to ANDV- and HTNV-infected endothelial cells results in the hyperphosphorylation of VEGFR2, while TULV infection failed to increase VEGFR2 phosphorylation. Concomitant with the VEGFR2 hyperphosphorylation, VE-cadherin was internalized to intracellular vesicles within ANDV- or HTNV-, but not TULV-, infected endothelial cells. Addition of angiopoietin-1 (Ang-1) or sphingosine-1-phosphate (S1P) to ANDV- or HTNV-infected cells blocked VE-cadherin internalization in response to VEGF. These findings are consistent with the ability of Ang-1 and S1P to inhibit hantavirus-induced endothelial cell permeability. Our results suggest that pathogenic hantaviruses disrupt fluid barrier properties of endothelial cell adherens junctions by enhancing VEGFR2-VE-cadherin pathway responses which increase paracellular permeability. These results provide a pathway-specific mechanism for the enhanced permeability of hantavirus-infected endothelial cells and suggest that stabilizing VE-cadherin within adherens junctions is a primary target for regulating endothelial cell permeability during pathogenic hantavirus infection.

T-cell immunoglobulin and mucin (TIM) contributes to the infection of human airway epithelial cells by pseudotype viruses containing Hantaan virus glycoproteins.

Hantaviruses are rodent-borne hemorrhagic fever viruses leading to serious diseases. Viral attachment and entry represent the first steps in virus transmission and are promising targets for antiviral therapeutic intervention. Here we investigated receptor use in human airway epithelium of the Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV). Using a biocontained recombinant vesicular stomatitis virus pseudotype platform, we provide first evidence for a role of the cellular phosphatidylserine (PS) receptors of the T-cell immunoglobulin and mucin (TIM) protein family in HTNV and ANDV infection. In line with previous studies, HTNV, but not ANDV, was able to use glycosaminoglycan heparan sulfate and αvß3 integrin as co-receptors. In sum, our studies demonstrate for the first time that hantaviruses make use of apoptotic mimicry for infection of human airway epithelium, which may explain why these viruses can easily break the species barrier.

Identification of oligopeptides mimicking the receptor-binding domain of Hantaan virus envelope glycoprotein from a phage-displayed peptide library.

Attachment of enveloped viruses to cells is triggered by the receptor-binding domain (RBD) on envelope glycoproteins (GP) binding to receptors located on the cell surface. To date, recognized receptors and RBD of hantaan virus (HTNV) have not been exactly defined. In this study, one monoclonal antibody (MAb) 3G1 possessing high neutralizing activity, which is directed against HTNV envelope glycoprotein G2, was used to determine the crucial motif of RBD. Peptide ligands binding to MAb 3G1 were selected from a 12 amino acid peptide library displayed on filamentous phages. After 3 rounds of selection, the binding capacity between phages and MAb 3G1 was examined byELISA. Afterwards the positive phage clones with high binding activity to MAb 3G1 were chosen and sequenced. The peptide sequences of positive phage clones were compared with that of HTNV 76-118 strain G2. A motif Y/F/WPW(X)HX1-2HY, aligned to the primary sequences of G2 96YPWHTAKCHY105, was identified from the peptide inserts in the 9 positive clones. Positive phages and synthesized peptide containing the motif were bound significantly to virus-susceptible cell (Vero-E6) membranes by ELISA and immunofluorescence assay, respectively. Therefore, the sequence on G2 between amino acid 96 and 105 may be a key motif of HTNV RBD recognized by viral receptors on target cell membranes. Further characterization of the motif would provide useful information in understanding of the cellular entry of HTNV.

High resolution cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly mechanisms.

Negative-strand RNA viruses condense their genome into helical nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of nucleocapsids usually prevents their full-length structural characterisation at high resolution. Here, we describe purification of full-length recombinant metastable helical nucleocapsid of Hantaan virus (Hantaviridae family, Bunyavirales order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerisation via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the nucleocapsid.

[Isolation and purification of host factors interacting with Hantaan virus nucleocapsid protein].

Objective To construct the plasmid expressing the fusion protein of Hantaan virus nucleocapsid protein (HTNV NP) with affinity tag, and isolate the host factors interacting with NP using the affinity purification. Methods The synthetic streptavidin-FLAG (SF) gene and HTNV NP gene were cloned into the mammalian eukaryotic expression vector to obtain the recombinant expression plasmid (pCAGGS-SF-NP). The plasmid pCAGGS-SF-NP was transfected into HEK293T cells, and the expression of SF-NP was detected by Western blotting. Next, cell lysates were mixed with StrepTrapTM HP agar beads. After incubating overnight at 4DegreesCelsius, the agar beads were transferred into affinity chromatography column and washed with elution buffer. Finally, the binding proteins that interacted with SF-NP were collected by competitive elution buffer with desthiobiotin, and then were subjected to SDS-PAGE. Results The recombinant SF-NP proteins were highly expressed in eukaryotic cells. The host factors interacting with SF-NP were successfully enriched by affinity purification, and confirmed by SDS-PAGE. Conclusion The host factors interacting with HTNV NP can be isolated by affinity purification.

In vivo characterization of the integrin beta3 as a receptor for Hantaan virus cellular entry.

Binding of viruses to cell surface molecules is an essential step in viral infection. In vitro studies suggested that the alpha(v)beta(3) integrin receptor is the epithelial cell receptor for Hantaan virus (HTNV). Whether beta(3) is in vivo the only or central cellular receptor for HTNV infection is not known. To investigate the role of beta(3) integrin for cellular entry of HTNV, we established an HTNV infection model in newborn murine pups. Infected pups died at an average age of 14.2 +/- 1.1 days with high levels of viral antigen detected in their brain, lung, and kidney. Pre-injection of blocking monoclonal antibodies (mAb) specific for either beta(3) or av prolonged survival significantly to a maximal average survival of 19.7 +/- 1.5 days (P <0.01) and 18.4 +/- 0.9 days (P < 0.01), respectively. XT-199, a chemical blocker of the alpha(v)beta(3) receptor also prolonged survival to 19.5 +/- 1.3 days (P < 0.01). In contrast to these receptor blockades, anti-HTNV antibody was not only able to prolong survival, but 20% of infected pups achieved long-term survival. An anti-murine beta(1) antibody comparatively prolonged survival (19.0 +/- 1.2 days), suggesting that HTNV infection is partly mediated through integrin beta(1) receptors as well as through beta(3) receptors in vivo. Our data demonstrate that the beta(3) receptor is important for HTNV infection in vivo, but also suggest that HTNV may utilize additional receptors beyond beta(3) for cellular entry within an organism.

Phosphorylation of the nucleocapsid protein of Hantaan virus by casein kinase II.

Hantaanvirus (HTNV) is the prototype of the genus Hantavirus, which belongs to the family Bunyaviridae. Hantaviruses are carried and transmitted by rodents and are known to cause two serious disease syndromes in humans i.e., hemorrhagic fever with renal syndrome (HFRS) and the hantavirus pulmonary syndrome (HPS). HTNV is an enveloped virus that contains a tripartite genome consisting of three negative-sense RNA segments (L, M, S), and the S and M segment of HTNV, respectively, encode the viral nucleocapsid protein (NP) and envelope glycoproteins. Possible phosphorylation motifs of casein kinase II (CKII) and protein kinase C (PKC) were identified in HTNV NP through bioinformatics searches. Sucrose gradient SDS-PAGE analysis indicated that dephosphorylated HTNV NP migrated faster than non-dephosphorylated NP, suggesting that HTNV NP is phosphorylated in infected Vero E6 cells. Immunoblot anaylsis of HTNV particles with anti-phosphoserine antibody and anti-phosphothreonine antibody after immunoprecipitation showed that viral particles are readily phosphorylated at threonine residues. In vitro kinase assay further showed that HTNV NP is phosphorylated by CK II, but not by PKC. Full length or truncated HTNV NPs expressed in E. coli were phosphorylated in vitro by CKII suggesting that phosphorylation may occur in vivo at multiple sites. Site specific mutagenesis studies suggest that HTNV NP phosphorylation might occur at unknown sites excluding the site-directly mutagenized locations. Taken together, HTNV NP can be phosphorylated mainly at threonine residues in vivo by CK II treatment.

Expression strategy of the M genome segment of Hantaan virus.

The medium (M) genome segment of Hantaan virus encodes the envelope glycoproteins, G1 and G2, in a continuous open reading frame with a gene order of 5'-G1-G2-3' with respect to the virus-complementary sense RNA. Because potential translation initiation codons and amino acids constituting typical signal sequences precede both the G1 and G2 genes, we sought to determine if G1 and G2 can be expressed independently. To investigate translational requirements for G1 and G2, we constructed M segment genes in which portions of the coding information were mutated or deleted, and transiently expressed these genes in eukaryotic cells by using a vaccinia virus/T7 RNA polymerase system. We found that G2 expression can occur by ribosomal access to the translation initiation codon preceding the G2 signal sequence (nucleotides 1934-1936), but that other upstream AUG codons cannot be used as efficiently. The presence of this codon, however, was not required for G2 expression because changing nucleotides 1934-1936 to CUG, GCG or AUG did not abrogate expression of G2. We also found that leaky ribosomal scanning, rather than internal initiation of translation was the most likely explanation for the observed independent translational initiation of G2, but that not all upstream, in-frame AUGs could serve as initiator codons. To assess the requirement for a continuous open reading frame for G1 and G2 expression, we expressed a gene which had G1 and G2 coding information in different reading frames. Although G1 was expressed at apparently normal levels, little or no G2 was expressed. In contrast, only G2 was expressed from a gene in which the carboxy-terminal G1 coding information was deleted and the remaining, truncated G1 was placed out of frame with respect to G2. These data suggest that reinitiation of translation may occur under some, but not all, circumstances when the polyprotein coding information is perturbed. Our results are consistent with biogenesis of G1 and G2 primarily or entirely according to the ribosomal scanning model.

Association of the nucleocapsid protein of the Seoul and Hantaan hantaviruses with small ubiquitin-like modifier-1-related molecules.

We performed yeast two-hybrid screening of a human kidney cell cDNA library to study the biological role of the hantavirus nucleocapsid protein (NP). We found that Seoul virus (SEOV) and Hantaan virus (HTNV) NPs were associated with small ubiquitin-like modifier (SUMO)-1-interacting proteins PIAS1, PIASxbeta, HIPK2, CHD3, and TTRAP, which interacted with the SUMO-1 conjugating enzyme (Ubc-9) and SUMO-1 in the yeast two-hybrid assay. Interactions between the HIPK2, CHD3, and TTRAP proteins and SEOV NP were also shown in a mammalian two-hybrid assay. However, there was no interaction between PIAS proteins and NP, which was probably due to the inhibitory effect of PIAS on transcription in the mammalian two-hybrid assay. Nevertheless, a co-expression experiment suggested the existence of a PIAS-NP interaction in the cytoplasm. The region spanning amino acids 100-125 of SEOV NP, which represents a critical region for NP-NP polymerization, was found to be responsible for the interaction with SUMO-1-related molecules in both the yeast and mammalian two-hybrid assays. These results add to the information on interactions of hantavirus NP and host cellular proteins.

Localization of Hantaan viral envelope glycoproteins by monoclonal antibodies in renal tissues from patients with Korean hemorrhagic fever H.

The role of viruses in several renal diseases is not documented clearly. The authors attempted to localize envelope glycoproteins of Hantaan virus in biopsy specimens from patients with Korean hemorrhagic fever (KHF) as evidence of direct viral invasion of renal tissues. The authors studied sequential sections of kidney biopsy specimens from 23 of 35 patients with serologically confirmed KHF diagnosed between June 1985 and December 1989. The sections were stained with the avidin-biotin-peroxidase complex method with monoclonal antibodies to G1 and G2 envelope glycoproteins. Control antibodies of the same isotype were used to rule out nonspecific staining, and hyperimmune rabbit sera or convalescent sera of patients with KHF were used for blocking tests. Normal renal tissues and kidney biopsy tissues from minimal-change nephrotic syndrome were used as negative control sections. The kidney biopsies were performed between the fifth and thirtieth days after onset of fever. The authors detected viral glycoproteins in renal tissues from 22 of the 23 patients. The viral glycoproteins were localized in the cytoplasm of the tubular epithelial cells, and the distribution of viral glycoproteins in the tubules was focal. Glycoproteins also were localized in the cytoplasm of the sloughed renal tubular epithelial cells, where tubular degenerative changes were prominent. These findings suggest the direct invasion of renal tubules by the virus and may partly explain the pathogenesis of acute renal failure in KHF.

Western blotting using recombinant Hantaan virus nucleocapsid protein expressed in silkworm as a serological confirmation of hantavirus infection in human sera.

Recombinant Hantaan virus nucleocapsid protein expressed in silkworm larvae was applied as a serological diagnostic antigen in Western blots (WB) of human sera. The sensitivity of this method was similar to that of the IFA test. Hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica diagnosed by their cross-reactivity in WB. The specificity of this method was higher than that of IFA test because the background was low. Sera that exhibited high background staining in the IFA test were readily diagnosed with this method. We recommended WB using recombinant Hantaan virus nucleocapsid antigen as a confirmatory procedure for the serodiagnosis of hantavirus.

Application of cDNA microarray technique to detection of gene expression in host cells infected with viruses.

cDNA microarray technique was used to monitor changes in mRNA levels in cells after Hantaan virus (HTNV) infection. The values of the ratio of medians for HTNV and Japanese encephalitis virus (JEV) at the early stage of infection were compared and found similar, suggesting that the same or similar genes are associated with the early events of infection with either virus. The reproducibility of values of the "ratio of medians" for HTNV was examined. We found that applying cluster analysis to the gene expression data groups efficiently together genes with the same function. Therefore, in analyzing the effects of viral infection on host cells by the cDNA microarray technique, clustering data appear to be necessary for gaining biological meaning from a dump of gene expression profiles obtained from virus-infected cells.

[The therapeutic effect of purified human leucocytic interferon-alpha on hemorrhagic fever with renal syndrome].

In order to know the therapeutic effect of purified human leucocytic interferon-alpha on hemorrhagic fever with renal syndrome diagnosed early (< or = 5 days). We treated 23 cases with clinical prognosis and antiviral effect significantly better than those of 21 cases in a random control group. The membrane protein (MP), nucleo protein (NP) in peripheral blood mononuclear cells (PBMC) and urine, the serum antibodies to MP and NP were determined before treatment and 2, 4, 6, 8, 10 days after the beginning of the treatment. The results showed that in the treatment group, the expressin of MP and NP in PBMC were significantly depressed. The rate of negative turning of MP and NP in PBMC was 65.22% (15/23) and 69.57% (16/23) in 10 days, while in the control group, it was 19.05% (4/21) and 14.29% (3/21). The rate of negative turning of MP and NP in urine were 78.57% (11/14) and 90.91% (10/11) and that in the control group was 30.00% (3/10) and 16.66% (1/6). There was a significant difference between the two groups (P < 0.01). The results indicated that IFN-alpha could attenuate the lesions caused directly by virus, improve patients condition and raise the cure rate through depressing the expression of the virus.

[Functional activity of hemagglutinins from Hantaan viruses strains and use of hemagglutinating antigens for serodiagnosis hemorrhagic fever with kidney syndrome]. / Funktsional'naia aktivnost' gemaggliutininov shtammov virusa Khantaan i ispol'zovanie gemaggliutiniruiushchikh antigenov dlia serodiagnostiki gemorrhagicheskoi likhoradki s pochechnym sindromom.

Hemagglutinins of Hantaan virus strains isolated in Southern Far East of Russia from patients with hemorrhagic fever with the renal syndrome and from rodents, natural vectors of the virus, were studied in the cross kinetic hemagglutination inhibition test with immune sera to the studied strains. The strains differed by the functional activity index. Use of hemagglutinins of functionally active Hantaan strains in serologic studies notably improves the diagnostic efficacy of the hemagglutination test in Hantavirus infection.

[Central hemodynamic disorders and rheological red blood cell properties in hemorrhagic fever patients with renal syndrome]. / Narusheniia tsentral'noi gemodinamiki i reologicheskikh svoistv éritrotsitov u bol'nykh gemorragicheskoi likhoradkoi s pochechnym sindromom.

The aim of this study was to detect a relationship between hemodynamic disorders in patients with hemorrhagic fever with the renal syndrome (HFRS) and erythrocyte aggregability and erythrocyte membrane ATPase activity. A total of 100 patients with HFRS of different severity were examined. Central hemodynamic parameters were studied: circulating blood volume, minute volume, cardiac index, stroke volume, and total peripheral vascular resistance during preoliguria, oliguria, and polyuria periods. Blood parameters were studied: percentage of minimum and maximum aggregation, disaggregation coefficient, activities of transport adenosine triphosphatases (Na, K, and Ca-activated ATPases and Mg-dependent ATPase). The main hemodynamic parameters were increased (p < 0.05) during early preoliguria and decreased during oliguria; during the polyuria period they again corresponded to the hyperkinetic circulation. The minimum erythrocyte aggregation increased by 110 and 130% in medium-severe and severe HFRS, respectively, the maximum erythrocyte aggregation by 20 and 28%, respectively (p < 0.05). Disaggregation coefficient decreased by 55%. The activities of Na, K(+)-ATPases decreased by 13% during preoliguria period, by 17.5% during oliguria, and by 11.7% during polyuria (p < 0.05) in patients with moderate disease. In severe disease these decreases were 14, 19, and 15%, respectively (p < 0.05). Similar changes were observed in the activities of Ca(++)-ATPase and Mg-dependent ATPase. Hence, the detected hemodynamic changes in patients with medium-severe and severe HFRS correlated with disorders in erythrocyte aggregability and decreased activity of transport ATPases, which can be used for evaluation of the severity of clinical condition and early diagnosis.