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.

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.

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.

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.

[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.

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.

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.

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.

Thrombocytopenia as a predictor of severe acute kidney injury in patients with Hantaan virus infections.

BACKGROUND: Hematological abnormalities often occur several days before kidney injury in patients with hemorrhagic fever with renal syndrome (HFRS). We aimed to investigate the prevalence and prognostic value of the early hematological markers in patients with HFRS caused by Hantaan virus (HTNV) infection. METHODS: In a retrospective cohort study, we analyzed the case records of 112 patients with acute HTNV infection and evaluated the hematological markers for early prediction and risk stratification of HFRS patients with acute kidney injury (AKI). RESULTS: Of 112 patients analyzed, 66 (59%) developed severe AKI, defined as either receipt of acute dialysis or increased serum creatinine ≥ 354 µmol/L. The prognostic accuracy of hematological markers, as quantified by the area under the receiver-operating-characteristic curve (AUC), was highest with the nadir platelet count (AUC, 0.89; 95% CI, 0.83-0.95), as compared with the admission platelet count (AUC, 0.84; 95% CI, 0.77-0.92), and the admission and peak leukocyte counts. The nadir platelet count correlated moderately with the levels of peak blood urea nitrogen (r = -0.616) and serum creatinine (r = -0.589), the length of hospital stay (r = -0.599), and the number of dialysis sessions that each patient received during hospital stay (r = -0.625). By multivariate analysis, decreased nadir platelet count remained independently associated with the development of severe AKI (odds ratio, 27.57; 95% CI, 6.96-109.16; P<0.0001). CONCLUSIONS: Thrombocytopenia, rather than leukocytosis, is independently associated with subsequent severe AKI among patients with acute HTNV 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.

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.

Gold nanoparticle enhanced immuno-PCR for ultrasensitive detection of Hantaan virus nucleocapsid protein.

A functionalized gold nanoparticle (GNP) enhanced ultrasensitive immuno-PCR assay (GNP-IPCR on ELISA plate), which was modified from the recent developed bio-barcode assay (BCA) technique, was developed to detect Hantaan virus nucleocapsid protein (HNP). During the assay, the target antigen HNP was captured by a polyclonal antibody coated on ELISA microplate wells, followed by adding GNP dually modified with oligonucleotides and a HNP specific monoclonal antibody L13 (mAb L13) to form a sandwich immuno-complex. The oligonucleotides on the GNP contained two strands: one as capture DNA immobilized on the surface of the GNP through Au-S bond and the other as signal amplification DNA, which was partially complementary with the capture DNA. After the immuno-complex was formed, the signal DNA was released by heating, and consequently characterized by PCR/gel electrophoresis and SYBR-Green real time PCR. The detection limit of this method could reach down to 10 fg/mL for detecting purified HNP in buffers as well as in human serum, which was approximately 7 orders of magnitude more sensitive than that of conventional ELISA. The current assay format might be adopted for other proteins that need ultra-high sensitive detection.

Hantaan virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon, interleukin-6 and tumor necrosis factor-alpha.

Hantaan virus (HTNV) infects endothelial cells and is associated with increased vascular permeability during hemorrhagic fever with renal syndrome (HFRS). The pattern of increased vascular permeability is mediated by immune response. Therefore, it is necessary to characterize the mechanism of HTNV involvement in the host's innate immune. In this study, the expression of five toll-like receptors (TLRs) was analyzed in Endothelial vein cells (EVC-304) following HTNV infection in vitro. TLR4 showed an altered expression after HTNV infection. HTNV infection significantly increased IFN-beta, IL-6 and TNF-alpha secretion from EVC-304 cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta, IL-6 and TNF-alpha production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HTNV-induced cytokine production. In conclusion, HTNV infection directly induces TLR4 expression and thereby enhanced production of IFN-beta, IL-6 and TNF-alpha, which may contribute to the host's innate immune response.

Use of Saccharomyces cerevisiae-expressed recombinant nucleocapsid protein to detect Hantaan virus-specific immunoglobulin G (IgG) and IgM in oral fluid.

Hantaan virus is the causative agent of severe hemorrhagic fever with renal syndrome. Clinical surveillance for Hantaan virus infection is unreliable, and laboratory verification is essential. The detection of virus-specific immunoglobulin M (IgM) and IgG in serum is most commonly used for the diagnosis of hantavirus infection. Testing of oral fluid samples instead of serum offers many advantages for surveillance. However, commercial tests for hantavirus-specific antibodies are unavailable. For the detection of Hantaan virus in the oral fluid of humans, we have developed a monoclonal antibody-based capture enzyme-linked immunosorbent IgM assay (IgM capture ELISA) and indirect enzyme-linked immunosorbent IgG and IgM assays (indirect IgG and IgM ELISAs) for paired serum and oral fluid samples using the Saccharomyces cerevisiae yeast-expressed nucleocapsid protein of the Hantaan-Fojnica virus. The sensitivity and specificity of the oral fluid IgM capture ELISA in comparison with the results of the serum Hantaan virus IgM assay were 96.7% and of 94.9%, respectively. Thus, data on the overall performance of the oral fluid IgM capture ELISA are in close agreement with those of the serum IgM assay, and the method exhibits the potential to serve as an easily transferable tool for large-scale epidemiological studies. Data on the indirect IgM ELISA also showed close agreement with the serum IgM assay data; however, the indirect IgG ELISA displayed a lower sensitivity and a lower specificity. In conclusion, the IgM capture ELISA can be used with oral fluid instead of serum samples for the diagnosis of Hantaan virus infection.

Ribavirin reveals a lethal threshold of allowable mutation frequency for Hantaan virus.

The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5'-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.

Degrons at the C terminus of the pathogenic but not the nonpathogenic hantavirus G1 tail direct proteasomal degradation.

Pathogenic hantaviruses cause two human diseases: hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). The hantavirus G1 protein contains a long, 142-amino-acid cytoplasmic tail, which in NY-1 virus (NY-1V) is ubiquitinated and proteasomally degraded (E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow, J. Virol. 77: 10760-10768, 2003). Here we report that the G1 cytoplasmic tails of pathogenic Andes (HPS) and Hantaan (HFRS) viruses are also degraded by the proteasome and that, in contrast, the G1 tail of nonpathogenic Prospect Hill virus (PHV) is stable and not proteasomally degraded. We determined that the signals which direct NY-1V G1 tail degradation are present in a hydrophobic region within the C-terminal 30 residues of the protein. In contrast to that of PHV, the NY-1V hydrophobic domain directs the proteasomal degradation of green fluorescent protein and constitutes an autonomous degradation signal, or "degron," within the NY-1V G1 tail. Replacing 4 noncontiguous residues of the NY-1V G1 tail with residues present in the stable PHV G1 tail resulted in a NY-1V G1 tail that was not degraded by the proteasome. In contrast, changing a different but overlapping set of 4 PHV residues to corresponding NY-1V residues directed proteasomal degradation of the PHV G1 tail. The G1 tails of pathogenic, but not nonpathogenic, hantaviruses contain intervening hydrophilic residues within the C-terminal hydrophobic domain, and amino acid substitutions that alter the stability or degradation of NY-1V or PHV G1 tails result from removing or adding intervening hydrophilic residues. Our results identify residues that selectively direct the proteasomal degradation of pathogenic hantavirus G1 tails. Although a role for the proteasomal degradation of the G1 tail in HPS or HFRS is unclear, these findings link G1 tail degradation to viral pathogenesis and suggest that degrons within hantavirus G1 tails are potential virulence determinants.

Activity of ribavirin against Hantaan virus correlates with production of ribavirin-5'-triphosphate, not with inhibition of IMP dehydrogenase.

Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5'-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5'-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 mug/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 mug/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.

Essential amino acids of the hantaan virus N protein in its interaction with RNA.

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

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.