Hantavirus-infected Cells Research Articles

Cryofixation of Inactivated Hantavirus-Infected Cells as a Method for Obtaining High-Quality Ultrastructural Preservation for Electron Microscopic Studies.

Hantaviruses rewire the host cell and induce extensive membrane rearrangements for their replication and the morphogenesis of the virion. Transmission electron microscopy (TEM) is a powerful technique for imaging these pathological membrane changes especially when combined with large volume electron tomography. Excellent preservation of membrane structure can be obtained when chemical fixation is combined with cryofixation via high pressure freezing making the samples amenable to serial-section tomographic reconstruction. Taking advantage of this, we have optimized a hybrid method that employs aldehyde fixation, a step that is essential for virus inactivation, followed by high-pressure freezing for ultrastructural study of Hantaan (HTN) and Andes (AND) virus infected Vero E6 cells. HTNV and ANDV are two species of the Orthohantavirus, from the Old and New World, respectively, and the causative agents of hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome in humans. We applied the method for the qualitative assessment of the perturbation of the endomembrane system induced by HTNV and ANDV in infected vs. mock-infected cells. Screening of serial-sections revealed consistency of membrane preservation across large volumes indicating potential of these samples for tomographic studies. Images revealed large-scale perturbations of the endomembrane system following HTNV-infection that included the dilation of the rough endoplasmic reticulum and fragmentation of the Golgi apparatus. Infected cells exhibited a tendency to accumulate large numbers of vacuoles that were especially apparent in ANDV. In summary, our hybrid method provides a path for the study of BSL-3 pathogens using cutting edge 3D-imaging technologies.

Hantavirus inhibits apoptosis by preventing mitochondrial membrane potential loss through up-regulation of the pro-survival factor BCL-2.

Hantaviruses, zoonotic RNA viruses belonging to the order Bunyavirales, cause two severe acute diseases in humans, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Hantavirus-infected patients show strong cytotoxic lymphocyte responses and hyperinflammation; however, infected cells remain mostly intact. Hantaviruses were recently shown to inhibit apoptosis in infected cells. By inhibiting granzyme B- and TRAIL-mediated apoptosis, hantaviruses specifically and efficiently inhibit cytotoxic lymphocyte-mediated killing of infected cells. Hantaviruses also strongly inhibit apoptosis triggered intrinsically; i.e., initiated through intracellular activation pathways different from those used by cytotoxic lymphocytes. However, insights into the latter mechanisms are currently largely unknown. Here, we dissected the mechanism behind how hantavirus infection, represented by the HFRS-causing Hantaan virus and the HPS-causing Andes virus, results in resistance to staurosporine-induced apoptosis. Less active caspase-8 and caspase-9, and consequently less active caspase-3, was observed in infected compared to uninfected staurosporine-exposed cells. While staurosporine-exposed uninfected cells showed massive release of pro-apoptotic cytochrome C into the cytosol, this was not observed in infected cells. Further, hantaviruses prevented activation of BAX and mitochondrial outer membrane permeabilization (MOMP). In parallel, a significant increase in levels of the pro-survival factor BCL-2 was observed in hantavirus-infected cells. Importantly, direct inhibition of BCL-2 by the inhibitor ABT-737, as well as silencing of BCL-2 by siRNA, resulted in apoptosis in staurosporine-exposed hantavirus-infected cells. Overall, we here provide a tentative mechanism by which hantaviruses protect infected cells from intrinsic apoptosis at the mitochondrial level by inducing an increased expression of the pro-survival factor BCL-2, thereby preventing MOMPs and subsequent activation of caspases. The variety of mechanisms used by hantaviruses to ensure survival of infected cells likely contribute to the persistent infection in natural hosts and may play a role in immunopathogenesis of HFRS and HPS in humans.

Hantavirus Inhibits TRAIL-Mediated Killing of Infected Cells by Downregulating Death Receptor 5

Cytotoxic lymphocytes normally kill virus-infected cells by apoptosis induction. Cytotoxic granule-dependent apoptosis induction engages the intrinsic apoptosis pathway, whereas death receptor (DR)-dependent apoptosis triggers the extrinsic apoptosis pathway. Hantaviruses, single-stranded RNA viruses of the order Bunyavirales, induce strong cytotoxic lymphocyte responses in infected humans. Cytotoxic lymphocytes, however, are largely incapable of eradicating hantavirus-infected cells. Here, we show that the prototypic hantavirus, Hantaan virus (HTNV), induces TRAIL production but strongly inhibits TRAIL-mediated extrinsic apoptosis induction in infected cells by downregulating DR5 cell surface expression. Mechanistic analyses revealed that HTNV triggers both 26S proteasome-dependent degradation of DR5 through direct ubiquitination of DR5 and hampers DR5 transport to the cell surface. These results corroborate earlier findings, demonstrating that hantavirus also inhibits cytotoxic cell granule-dependent apoptosis induction. Together, these findings show that HTNV counteracts intrinsic and extrinsic apoptosis induction pathways, providing a defense mechanism utilized by hantaviruses to inhibit cytotoxic cell-mediated eradication of infected cells.

Lentivirus Expression of Hantavirus Nucleocapsid Proteins

Hantaviruses are classified as category A pathogens due to the high mortality rate and potential for major health impact. Therefore, virus propagation is restricted to the Biological Safety Level-3 containment laboratory. In this work, we utilized the recombinant lentivirus delivery system which can be used in Biological Safety Level-2 containment. Recombinant lentiviruses were obtained using Gateway cloning technology. Lentiviruses containing S segment of Hantaan, Sin Nombre, Prospect Hill, or Andes viruses were used to transduce lung carcinoma epithelial type II cells (A549). Accumulation of hantavirus S segment RNA was detected using real-time PCR. Additionally, expression of hantavirus nucleocapsid protein was detected in cells transduced with lentiviruses coding for hantavirus S segment. Hantavirus N proteins were found localized in perinuclear region. Interestingly, localization of N protein was similar to that found in hantavirus infected cells. Therefore, we suggest that lentivirus delivery approach could be used to study the mechanisms of hantavirus pathogenesis in Biological Safety Level-2 containment.

NK cell activation in human hantavirus infection explained by virus-induced IL-15/IL15Rα expression.

Clinical infection with hantaviruses cause two severe acute diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases are characterized by strong immune activation, increased vascular permeability, and up to 50% case-fatality rates. One prominent feature observed in clinical hantavirus infection is rapid expansion of natural killer (NK) cells in peripheral blood of affected individuals. We here describe an unusually high state of activation of such expanding NK cells in the acute phase of clinical Puumala hantavirus infection. Expanding NK cells expressed markedly increased levels of activating NK cell receptors and cytotoxic effector molecules. In search for possible mechanisms behind this NK cell activation, we observed virus-induced IL-15 and IL-15Rα on infected endothelial and epithelial cells. Hantavirus-infected cells were shown to strongly activate NK cells in a cell-cell contact-dependent way, and this response was blocked with anti-IL-15 antibodies. Surprisingly, the strength of the IL-15-dependent NK cell response was such that it led to killing of uninfected endothelial cells despite expression of normal levels of HLA class I. In contrast, hantavirus-infected cells were resistant to NK cell lysis, due to a combination of virus-induced increase in HLA class I expression levels and hantavirus-mediated inhibition of apoptosis induction. In summary, we here describe a possible mechanism explaining the massive NK cell activation and proliferation observed in HFRS patients caused by Puumala hantavirus infection. The results add further insights into mechanisms behind the immunopathogenesis of hantavirus infections in humans and identify new possible targets for intervention.

Andes virus nucleocapsid protein interrupts protein kinase R dimerization to counteract host interference in viral protein synthesis.

Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the manufacture of viral proteins. The studies reported here reveal that the hantavirus nucleocapsid protein counteracts the PKR antiviral response by inhibiting PKR dimerization, which is required for its activation. We report the discovery of a new PKR inhibitor whose expression in hantavirus-infected cells prevents the PKR-induced host translational shutdown to ensure the continuous synthesis of viral proteins required for efficient virus replication.

The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon‐beta promoter

The S RNA genome segment of hantaviruses carried by Arvicolinae and Sigmodontinae rodents encodes the nucleocapsid (N) protein and has an overlapping (+1) open reading frame (ORF) for a putative nonstructural protein (NSs). The aim of this study was to determine whether the ORF is functional. A protein corresponding to the predicted size of Tula virus (TULV) NSs was detected using coupled in vitro transcription and translation from a cloned S segment cDNA, and a protein corresponding to the predicted size of Puumala virus (PUUV) NSs was detected in infected cells by Western blotting with an anti-peptide serum. The activities of the interferon beta (IFN-beta) promoter, and nuclear factor kappa B (NF-kappaB)- and interferon regulatory factor-3 (IRF-3) responsive promoters, were inhibited in COS-7 cells transiently expressing TULV or PUUV NSs. Also IFN-beta mRNA levels in IFN-competent MRC5 cells either infected with TULV or transiently expressing NSs were decreased. These data demonstrate that Tula and Puumala hantaviruses have a functional NSs ORF. The findings may explain why the NSs ORF has been preserved in the genome of most hantaviruses during their long evolution and why hantavirus-infected cells secrete relatively low levels of IFNs.

Andes virus stimulates interferon‐inducible MxA protein expression in endothelial cells

MxA is a cellular protein activated in cells treated with type I interferons. MxA protein is a member of the dynamin superfamily of large guanosine triphosphatases. A unique property of Mx1 GTPases is their antiviral activity against a wide range of RNA viruses. Replication of several hantavirus strains is inhibited in cells constitutively expressing MxA protein. We have found that Andes virus (ANDV) infection up regulates transcription of MxA RNA and expression of MxA protein in human endothelial cells in vitro. Activation of MxA gene expression requires virus replication. Up-regulation of MxA gene expression in hantavirus infected cells varies depending on the cell type. The degree of activation of MxA gene expression is inversely correlated with the efficiency of hantavirus replication. Additionally, MxA protein is co-localized with hantavirus nucleocapsid protein in infected cell. Our data suggest that MxA by interacting with the virus nucleocapsid protein inhibits production of new infectious virus particles.

Development and application of protein G antibody assay for the detection of antibody to hantavirus

A new serodiagnostic method designated protein-G antibody assay (PGA) was developed for detection of hantavirus infection in various species of animals. The assay procedure includes reacting the sera with hantavirusinfected cells on glass slides, followed by incubation of biotinylated protein G and amplification with the avidin-biotinylated peroxidase complex. Specific antibody in rabbit, rat, mouse and Mongolian gerbil serum was detected by this method. The PGA titres were similar to those of the neutralization titre. In the sera of Mongolian gerbils infected with strain SR-11, antibody was first detected 10 days post infection, and the titre increased to 1:256 at 18 days post-infection. PGA was evaluated using sera of urban rats ( Rattus norvegicns) captured in an endemic area of hantavirus infection. The negative (⩽ 1:1, 24 62 , 38.7%) and positive groups (⩾1:16, 38 62 , 61.3%) were clearly distinguished. PGA titres were closely related to IFA titres in the sera. Two of 10 sera from Clethrionomys rufocanus and one from Apodemus speciosus captured in the same endemic area were positive to both PGA and IFA. These data indicate that PGA is a simple and useful method for seroepizootiological surveys of hantavirus infection, especially in wild rodent reservoirs.

Morphology and morphogenesis of viruses of hemorrhagic fever with renal syndrome. II. Inclusion bodies--ultrastructural markers of hantavirus-infected cells.

Three distinctive types of viral inclusion bodies were identified in hantavirus-infected cells by thin-section electron microscopy. The inclusion bodies, designated granular, granulofilamentous, and filamentous, were intracytoplasmic and closely associated with the rough endoplasmic reticulum and Golgi cisternae. Virus specificity of the inclusions was verified by immune colloidal gold and immunoperoxidase labeling. The inclusion bodies were a common morphological marker for 13 strains of hantaviruses studied, irrespective of their origin.