Hantavirus Entry Research Articles

Dihydropyridine-derived calcium channel blocker as a promising anti-hantavirus entry inhibitor.

Hantaviruses, the causative agent for two types of hemorrhagic fevers, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), are distributed from Eurasia to America. HFRS and HPS have mortality rates of up to 15% or 45%, respectively. Currently, no certified therapeutic has been licensed to treat hantavirus infection. In this study, we discovered that benidipine hydrochloride, a calcium channel blocker, inhibits the entry of hantaviruses in vitro. Moreover, an array of calcium channel inhibitors, such as cilnidipine, felodipine, amlodipine, manidipine, nicardipine, and nisoldipine, exhibit similar antiviral properties. Using pseudotyped vesicular stomatitis viruses harboring the different hantavirus glycoproteins, we demonstrate that benidipine hydrochloride inhibits the infection by both HFRS- and HPS-causing hantaviruses. The results of our study indicate the possibility of repurposing FDA-approved calcium channel blockers for the treatment of hantavirus infection, and they also indicate the need for further research in vivo.

Genetic depletion studies inform receptor usage by virulent hantaviruses in human endothelial cells.

Hantaviruses are RNA viruses with known epidemic threat and potential for emergence. Several rodent-borne hantaviruses cause zoonoses accompanied by severe illness and death. However, assessments of zoonotic risk and the development of countermeasures are challenged by our limited knowledge of the molecular mechanisms of hantavirus infection, including the identities of cell entry receptors and their roles in influencing viral host range and virulence. Despite the long-standing presumption that β3/β1-containing integrins are the major hantavirus entry receptors, rigorous genetic loss-of-function evidence supporting their requirement, and that of decay-accelerating factor (DAF), is lacking. Here, we used CRISPR/Cas9 engineering to knockout candidate hantavirus receptors, singly and in combination, in a human endothelial cell line that recapitulates the properties of primary microvascular endothelial cells, the major targets of viral infection in humans. The loss of β3 integrin, β1 integrin, and/or DAF had little or no effect on entry by a large panel of hantaviruses. By contrast, loss of protocadherin-1, a recently identified entry receptor for some hantaviruses, substantially reduced hantavirus entry and infection. We conclude that major host molecules necessary for endothelial cell entry by PCDH1-independent hantaviruses remain to be discovered.

Identification of Novel Antiviral Compounds Targeting Entry of Hantaviruses.

Hemorrhagic fever viruses, among them orthohantaviruses, arenaviruses and filoviruses, are responsible for some of the most severe human diseases and represent a serious challenge for public health. The current limited therapeutic options and available vaccines make the development of novel efficacious antiviral agents an urgent need. Inhibiting viral attachment and entry is a promising strategy for the development of new treatments and to prevent all subsequent steps in virus infection. Here, we developed a fluorescence-based screening assay for the identification of new antivirals against hemorrhagic fever virus entry. We screened a phytochemical library containing 320 natural compounds using a validated VSV pseudotype platform bearing the glycoprotein of the virus of interest and encoding enhanced green fluorescent protein (EGFP). EGFP expression allows the quantitative detection of infection and the identification of compounds affecting viral entry. We identified several hits against four pseudoviruses for the orthohantaviruses Hantaan (HTNV) and Andes (ANDV), the filovirus Ebola (EBOV) and the arenavirus Lassa (LASV). Two selected inhibitors, emetine dihydrochloride and tetrandrine, were validated with infectious pathogenic HTNV in a BSL-3 laboratory. This study provides potential therapeutics against emerging virus infection, and highlights the importance of drug repurposing.

Macropinocytosis contributes to hantavirus entry into human airway epithelial cells

Hantaviruses are emerging rodent-borne negative-strand RNA viruses associated with severe human diseases. Zoonotic transmission occurs via aerosols of contaminated rodent excreta and cells of the human respiratory epithelium represent likely early targets. Here we investigated cellular factors involved in entry of the pathogenic Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV) into human respiratory epithelial cells. Screening of a kinase inhibitor library using a biocontained recombinant vesicular stomatitis virus pseudotype platform revealed differential requirement for host kinases for HTNV and ANDV entry and provided first hints for an involvement of macropinocytosis. Examination of a selected panel of well-defined inhibitors of endocytosis confirmed that both HTNV and ANDV enter human respiratory epithelial cells via a pathway that critically depends on sodium proton exchangers and actin, hallmarks of macropinocytosis. However, HTNV and ANDV differed in their individual requirements for regulatory factors of macropinocytosis, indicating virus-specific differences.

Two Point Mutations in Old World Hantavirus Glycoproteins Afford the Generation of Highly Infectious Recombinant Vesicular Stomatitis Virus Vectors.

Rodent-to-human transmission of hantaviruses is associated with severe disease. Currently, no FDA-approved, specific antivirals or vaccines are available, and the requirement for high biocontainment (biosafety level 3 [BSL-3]) laboratories limits hantavirus research. To study hantavirus entry in a BSL-2 laboratory, we set out to generate replication-competent, recombinant vesicular stomatitis viruses (rVSVs) bearing the Gn and Gc (Gn/Gc) entry glycoproteins. As previously reported, rVSVs bearing New World hantavirus Gn/Gc were readily rescued from cDNAs, but their counterparts bearing Gn/Gc from the Old World hantaviruses, Hantaan virus (HTNV) or Dobrava-Belgrade virus (DOBV), were refractory to rescue. However, serial passage of the rescued rVSV-HTNV Gn/Gc virus markedly increased its infectivity and capacity for cell-to-cell spread. This gain in viral fitness was associated with the acquisition of two point mutations: I532K in the cytoplasmic tail of Gn and S1094L in the membrane-proximal stem of Gc. Follow-up experiments with rVSVs and single-cycle VSV pseudotypes confirmed these results. Mechanistic studies revealed that both mutations were determinative and contributed to viral infectivity in a synergistic manner. Our findings indicate that the primary mode of action of these mutations is to relocalize HTNV Gn/Gc from the Golgi complex to the cell surface, thereby affording significantly enhanced Gn/Gc incorporation into budding VSV particles. Finally, I532K/S1094L mutations in DOBV Gn/Gc permitted the rescue of rVSV-DOBV Gn/Gc, demonstrating that incorporation of cognate mutations into other hantaviral Gn/Gc proteins could afford the generation of rVSVs that are otherwise challenging to rescue. The robust replication-competent rVSVs, bearing HTNV and DOBV Gn/Gc, reported herein may also have utility as vaccines.IMPORTANCE Human hantavirus infections cause hantavirus pulmonary syndrome in the Americas and hemorrhagic fever with renal syndrome (HFRS) in Eurasia. No FDA-approved vaccines and therapeutics exist for these deadly viruses, and their development is limited by the requirement for high biocontainment. In this study, we identified and characterized key amino acid changes in the surface glycoproteins of HFRS-causing Hantaan virus that enhance their incorporation into recombinant vesicular stomatitis virus (rVSV) particles. The replication-competent rVSVs encoding Hantaan virus and Dobrava-Belgrade virus glycoproteins described in this work provide a powerful and facile system to study hantavirus entry under lower biocontainment and may have utility as hantavirus vaccines.

Hantavirus entry: Perspectives and recent advances.

Hantaviruses are important zoonotic pathogens of public health importance that are found on all continents except Antarctica and are associated with hemorrhagic fever with renal syndrome (HFRS) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Despite the significant disease burden they cause, no FDA-approved specific therapeutics or vaccines exist against these lethal viruses. The lack of available interventions is largely due to an incomplete understanding of hantavirus pathogenesis and molecular mechanisms of virus replication, including cellular entry. Hantavirus Gn/Gc glycoproteins are the only viral proteins exposed on the surface of virions and are necessary and sufficient to orchestrate virus attachment and entry. In vitro studies have implicated integrins (β1-3), DAF/CD55, and gC1qR as candidate receptors that mediate viral attachment for both Old World and New World hantaviruses. Recently, protocadherin-1 (PCDH1) was demonstrated as a requirement for cellular attachment and entry of New World hantaviruses in vitro and lethal HPS in vivo, making it the first clade-specific host factor to be identified. Attachment of hantavirus particles to cellular receptors induces their internalization by clathrin-mediated, dynamin-independent, or macropinocytosis-like mechanisms, followed by particle trafficking to an endosomal compartment where the fusion of viral and endosomal membranes can occur. Following membrane fusion, which requires cholesterol and acid pH, viral nucleocapsids escape into the cytoplasm and launch genome replication. In this review, we discuss the current mechanistic understanding of hantavirus entry, highlight gaps in our existing knowledge, and suggest areas for future inquiry.

Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

The Major Cellular Sterol Regulatory Pathway Is Required for Andes Virus Infection

The Bunyaviridae comprise a large family of RNA viruses with worldwide distribution and includes the pathogenic New World hantavirus, Andes virus (ANDV). Host factors needed for hantavirus entry remain largely enigmatic and therapeutics are unavailable. To identify cellular requirements for ANDV infection, we performed two parallel genetic screens. Analysis of a large library of insertionally mutagenized human haploid cells and a siRNA genomic screen converged on components (SREBP-2, SCAP, S1P and S2P) of the sterol regulatory pathway as critically important for infection by ANDV. The significance of this pathway was confirmed using functionally deficient cells, TALEN-mediated gene disruption, RNA interference and pharmacologic inhibition. Disruption of sterol regulatory complex function impaired ANDV internalization without affecting virus binding. Pharmacologic manipulation of cholesterol levels demonstrated that ANDV entry is sensitive to changes in cellular cholesterol and raises the possibility that clinically approved regulators of sterol synthesis may prove useful for combating ANDV infection.

Plasma Levels of Soluble Urokinase-Type Plasminogen Activator Receptor Associate with the Clinical Severity of Acute Puumala Hantavirus Infection

ObjectivesUrokinase-type plasminogen activator receptor is a multifunctional glycoprotein, the expression of which is increased during inflammation. It is known to bind to β3-integrins, which are elementary for the cellular entry of hantaviruses. Plasma soluble form of the receptor (suPAR) levels were evaluated as a predictor of severe Puumala hantavirus (PUUV) infection and as a possible factor involved in the pathogenesis of the disease.DesignA single-centre prospective cohort study.Subjects and MethodsPlasma suPAR levels were measured twice during the acute phase and once during the convalescence in 97 patients with serologically confirmed acute PUUV infection using a commercial enzyme-linked immunosorbent assay (ELISA).ResultsThe plasma suPAR levels were significantly higher during the acute phase compared to the control values after the hospitalization (median 8.7 ng/ml, range 4.0–18.2 ng/ml vs. median 4.7 ng/ml, range 2.4–12.2 ng/ml, P<0.001). The maximum suPAR levels correlated with several variables reflecting the severity of the disease. There was a positive correlation with maximum leukocyte count (r = 0.475, p<0.001), maximum plasma creatinine concentration (r = 0.378, p<0.001), change in weight during the hospitalization (r = 0.406, p<0.001) and the length of hospitalization (r = 0.325, p = 0.001), and an inverse correlation with minimum platelet count (r = −0.325, p = 0.001) and minimum hematocrit (r = −0.369, p<0.001).ConclusionPlasma suPAR values are markedly increased during acute PUUV infection and associate with the severity of the disease. The overexpression of suPAR possibly activates β3-integrin in PUUV infection, and thus might be involved in the pathogenesis of the disease.

Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β 3 -Integrin Receptor

Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the alpha(v)beta(3) integrin, and transfection of human beta(3) integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-beta(3) antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified alpha(v)beta(3), we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 mug/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use beta(3) integrins and PHV uses a different receptor, beta1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via beta(3) integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

New Ecological Aspects of Hantavirus Infection: A Change of A Paradigm and a Challenge of Prevention- A Review

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.

Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins.

Hantaviruses replicate primarily in the vascular endothelium and cause two human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). In this report, we demonstrate that the cellular entry of HFRS-associated hantaviruses is facilitated by specific integrins expressed on platelets, endothelial cells, and macrophages. Infection of human umbilical vein endothelial cells and Vero E6 cells by the HFRS-causing hantaviruses Hantaan (HTN), Seoul (SEO), and Puumala (PUU) is inhibited by antibodies to alphavbeta3 integrins and by the integrin ligand vitronectin. The cellular entry of HTN, SEO, and PUU viruses, but not the nonpathogenic Prospect Hill (PH) hantavirus (i.e., a virus with no associated human disease), was also mediated by introducting recombinant alphaIIbbeta3 or alphavbeta3 integrins into beta3-integrin-deficient CHO cells. In addition, PH infectivity was not inhibited by alphavbeta3-specific sera or vitronectin but was blocked by alpha5beta1-specific sera and the integrin ligand fibronectin. RGD tripeptides, which are required for many integrin-ligand interactions, are absent from all hantavirus G1 and G2 surface glycoproteins, and GRGDSP peptides did not inhibit hantavirus infectivity. Further, a mouse-human hybrid beta3 integrin-specific Fab fragment, c7E3 (ReoPro), also inhibited the infectivity of HTN, SEO, and PUU as well as HPS-associated hantaviruses, Sin Nombre (SN) and New York-1 (NY-1). These findings indicate that pathogenic HPS- and HFRS-causing hantaviruses enter cells via beta3 integrins, which are present on the surfaces of platelets, endothelial cells, and macrophages. Since beta3 integrins regulate vascular permeability and platelet function, these findings also correlate beta3 integrin usage with common elements of hantavirus pathogenesis.

Beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure.

Newly emerged hantaviruses replicate primarily in the pulmonary endothelium, cause acute platelet loss, and result in hantavirus pulmonary syndrome (HPS). We now report that specific integrins expressed on platelets and endothelial cells permit the cellular entry of HPS-associated hantaviruses. Infection with HPS-associated hantaviruses, NY-1 and Sin Nombre virus (SNV), is inhibited by antibodies to beta3 integrins and by the beta3-integrin ligand, vitronectin. In contrast, infection with the nonpathogenic (no associated human disease) Prospect Hill virus was inhibited by fibronectin and beta1-specific antibodies but not by beta3-specific antibodies or vitronectin. Transfection with recombinant alphaIIb beta3 or alphav beta3 integrins rendered cells permissive to NY-1 and SNV but not Prospect Hill virus infection, indicating that alphaIIb beta3 and alphav beta3 integrins mediate the entry of NY-1 and SNV hantaviruses. Furthermore, entry is divalent cation independent, not blocked by arginine-glycine-aspartic acid peptides and still mediated by, ligand-binding defective, alphaIIb beta3-integrin mutants. Hence, NY-1 and SNV entry is independent of beta3 integrin binding to physiologic ligands. These findings implicate integrins as cellular receptors for hantaviruses and indicate that hantavirus pathogenicity correlates with integrin usage.