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The SARS-CoV-2 Omicron variant and its sublineages continue to cause COVID-19-associated pediatric hospitalizations, severe disease, and death globally. BNT162b2 and CoronaVac are the main vaccines used in Chile. Much less is known about the Wuhan-Hu-1 strain-based vaccines in the pediatric population compared to adults. Given the worldwide need for booster vaccinations to stimulate the immune response against new Omicron variants of SARS-CoV-2, we characterized the humoral and cellular immune response against Omicron variant BA.1 in a pediatric cohort aged 10 to 16 years who received heterologous vaccination based on two doses of CoronaVac, two doses of CoronaVac (2x) plus one booster dose of BNT162b2 [CoronaVac(2x) + BNT162b2 (1x)], two doses of CoronaVac plus two booster doses of BNT162b2 [CoronaVac(2x) + BNT162b2 (2x)], and three doses of BNT162b2. We observed that the [CoronaVac(2x) + BNT162b2 (2x)] vaccination showed higher anti-S1 and neutralizing antibody titers and CD4 and CD8 T cell immunity specific to the Omicron variant compared to immunization with two doses of CoronaVac alone. Furthermore, from all groups tested, immunity against Omicron was highest in individuals who received three doses of BNT162b2. We conclude that booster vaccination with BNT162b2, compared to two doses of CoronaVac alone, induces a greater protective immunity.
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Hantaviridae is a family for negative-sense RNA viruses with genomes of about 10.5-14.6 kb. These viruses are maintained in and/or transmitted by fish, reptiles, and mammals. Several orthohantaviruses can infect humans, causing mild, severe, and sometimes-fatal diseases. Hantavirids produce enveloped virions containing three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hantaviridae, which is available at ictv.global/report/hantaviridae.
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Vírus de RNA , Animais , Humanos , Vírus de RNA de Sentido Negativo , Vírion/genética , Nucleoproteínas , Fases de Leitura Aberta , MamíferosRESUMO
A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.
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The official classification of newly discovered or long-known unassigned viruses by the International Committee on Taxonomy of Viruses (ICTV) requires the deposition of coding-complete or -near-complete virus genome sequences in GenBank to fulfill a requirement of the taxonomic proposal (TaxoProp) process. However, this requirement is fairly new; thus, genomic sequence information is fragmented or absent for many already-classified viruses. As a result, taxon-wide modern phylogenetic analyses are often challenging, if not impossible. This problem is particularly eminent among viruses with segmented genomes, such as bunyavirals, which were frequently classified solely based on single-segment sequence information. To solve this issue for one bunyaviral family, Hantaviridae, we call on the community to provide additional sequence information for incompletely sequenced classified viruses by mid-June 2023. Such sequence information may be sufficient to prevent their possible declassification during the ongoing efforts to establish a coherent, consistent, and evolution-based hantavirid taxonomy.
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Vírus de RNA , Vírus , Filogenia , Vírus/genética , Genômica , Bases de Dados de Ácidos NucleicosRESUMO
BACKGROUND: A major challenge of the SARS-CoV-2 pandemic is to better define "protective thresholds" to guide the global response. We aimed to characterize the longitudinal dynamics of the antibody responses in naturally infected individuals in Chile and compared them to humoral responses induced after immunization with CoronaVac-based on an inactivated whole virus -or the BNT162b2- based on mRNA-vaccines. We also contrasted them with the respective effectiveness and efficacy data available for both vaccines. METHODS: We determined and compared the longitudinal neutralizing (nAb) and anti-nucleocapsid (anti-N) antibody responses of 74 COVID-19 individuals (37 outpatient and 37 hospitalized) during the acute disease and convalescence. We also assessed the antibody boosting of 36 of these individuals who were immunized after convalescence with either the CoronaVac (n = 30) or the BNT162b2 (n = 6) vaccines. Antibody titres were also measured for 50 naïve individuals immunized with two doses of CoronaVac (n = 35) or BNT162b2 (n = 15) vaccines. The neutralizing level after vaccination was compared to those of convalescent individuals and the predicted efficacy was estimated. FINDINGS: SARS-CoV-2 infection induced robust nAb and anti-N antibody responses lasting >9 months, but showing a rapid nAb decay. After convalescence, nAb titres were significantly boosted by vaccination with CoronaVac or BNT162b2. In naïve individuals, the calculated mean titre induced by two doses of CoronaVac or BNT162b2 was 0·2 times and 5.2 times, respectively, that of convalescent individuals, which has been proposed as threshold of protection. CoronaVac induced no or only modest anti-N antibody responses. Using two proposed logistic models, the predicted efficacy of BNT162b2 was estimated at 97%, in close agreement with phase 3 efficacy studies, while for CoronaVac it was â¼50% corresponding to the lowest range of clinical trials and below the real-life data from Chile (from February 2 through May 1, 2021 during the predominant circulation of the Gamma variant), where the estimated vaccine effectiveness to prevent COVID-19 was 62·8-64·6%. INTERPRETATION: The decay of nAbs titres in previously infected individuals over time indicates that vaccination is needed to boost humoral memory responses. Immunization of naïve individuals with two doses of CoronaVac induced nAbs titres that were significantly lower to that of convalescent patients, and similar to vaccination with one dose of BTN162b2. The real life effectiveness for CoronaVac in Chile was higher than estimated; indicating that lower titres and additional cellular immune responses induced by CoronaVac might afford protection in a highly immunized population. Nevertheless, the lower nAb titre induced by two doses of CoronaVac as compared to the BTN162b2 vaccine in naïve individuals, highlights the need of booster immunizations over time to maintain protective levels of antibody, particularly with the emergence of new SARS-CoV-2 variants. FUNDING: FONDECYT 1161971, 1212023, 1181799, FONDECYT Postdoctorado 3190706 and 3190648, ANID Becas/Doctorado Nacional 21212258, PIA ACT 1408, CONICYT REDES180170, Centro Ciencia & Vida, FB210008, Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia grants from the Agencia Nacional de Investigación y Desarrollo (ANID) of Chile; NIH-NIAD grants U19AI135972, R01AI132633 and contracts HHSN272201400008C and 75N93019C00051; the JPB Foundation, the Open Philanthropy Project grant 2020-215611 (5384); and by anonymous donors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacina BNT162 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Convalescença , HumanosRESUMO
(1) Background: Haloarchaea comprise extremely halophilic organisms of the Archaea domain. They are single-cell organisms with distinctive membrane lipids and a protein-based cell wall or surface layer (S-layer) formed by a glycoprotein array. Pleolipoviruses, which infect haloarchaeal cells, have an envelope analogous to eukaryotic enveloped viruses. One such member, Halorubrum pleomorphic virus 6 (HRPV-6), has been shown to enter host cells through virus-cell membrane fusion. The HRPV-6 fusion activity was attributed to its VP4-like spike protein, but the physiological trigger required to induce membrane fusion remains yet unknown. (2) Methods: We used SDS-PAGE mass spectroscopy to characterize the S-layer extract, established a proteoliposome system, and used R18-fluorescence dequenching to measure membrane fusion. (3) Results: We show that the S-layer extraction by Mg2+ chelating from the HRPV-6 host, Halorubrum sp. SS7-4, abrogates HRPV-6 membrane fusion. When we in turn reconstituted the S-layer extract from Hrr. sp. SS7-4 onto liposomes in the presence of Mg2+, HRPV-6 membrane fusion with the proteoliposomes could be readily observed. This was not the case with liposomes alone or with proteoliposomes carrying the S-layer extract from other haloarchaea, such as Haloferax volcanii. (4) Conclusions: The S-layer extract from the host, Hrr. sp. SS7-4, corresponds to the physiological fusion trigger of HRPV-6.
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Proteínas Arqueais/metabolismo , Vírus de Archaea/fisiologia , Halorubrum/virologia , Glicoproteínas de Membrana/metabolismo , Internalização do Vírus , Vírus de Archaea/ultraestrutura , Halorubrum/ultraestrutura , Interações entre Hospedeiro e Microrganismos , Fusão de Membrana , Proteolipídeos/metabolismoRESUMO
The durability of circulating neutralizing antibody (nAb) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their boosting by vaccination remains to be defined. We show that outpatient and hospitalized SARS-CoV-2 seropositive individuals mount a robust neutralizing antibody (nAb) response that peaks at days 23 and 27 post-symptom onset, respectively. Although nAb titers remained higher in hospitalized patients, both study groups showed long-lasting nAb responses that can persist for up to 12 months after natural infection. These nAb responses in previously seropositive individuals can be significantly boosted through immunization with two doses of the CoronaVac (Sinovac) or one dose of the BNT162b2 (BioNTech/Pfizer) vaccines, suggesting a substantial induction of B cell memory responses. Noteworthy, three obese previously seropositive individuals failed to mount a booster response upon vaccination, warranting further studies in this population. Immunization of naïve individuals with two doses of the CoronaVac vaccine or one dose of the BNT162b2 vaccine elicited similar levels of nAbs compared to seropositive individuals 4.2 to 13.3 months post-infection with SARS-CoV-2. Thus, this preliminary evidence suggests that both, seropositive and naïve individuals, require two doses of CoronaVac to ensure the induction of robust nAb titers.
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Phenuiviridae is a large family of arthropod-borne viruses with over 100 species worldwide. Several cause severe diseases in both humans and livestock. Global warming and the apparent geographical expansion of arthropod vectors are good reasons to seriously consider these viruses potential agents of emerging diseases. With an increasing frequency and number of epidemics, some phenuiviruses represent a global threat to public and veterinary health. This review focuses on the early stage of phenuivirus infection in mammalian host cells. We address current knowledge on each step of the cell entry process, from virus binding to penetration into the cytosol. Virus receptors, endocytosis, and fusion mechanisms are discussed in light of the most recent progress on the entry of banda-, phlebo-, and uukuviruses, which together constitute the three prominent genera in the Phenuiviridae family.
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Infecções por Bunyaviridae/virologia , Mamíferos/virologia , Phlebovirus/fisiologia , Internalização do Vírus , Animais , Infecções por Bunyaviridae/fisiopatologia , Endocitose , Humanos , Phlebovirus/genética , Ligação ViralRESUMO
Hantaviruses are rodent-borne viruses causing serious zoonotic outbreaks worldwide for which no treatment is available. Hantavirus particles are pleomorphic and display a characteristic square surface lattice. The envelope glycoproteins Gn and Gc form heterodimers that further assemble into tetrameric spikes, the lattice building blocks. The glycoproteins, which are the sole targets of neutralizing antibodies, drive virus entry via receptor-mediated endocytosis and endosomal membrane fusion. Here we describe the high-resolution X-ray structures of the heterodimer of Gc and the Gn head and of the homotetrameric Gn base. Docking them into an 11.4-Å-resolution cryoelectron tomography map of the hantavirus surface accounted for the complete extramembrane portion of the viral glycoprotein shell and allowed a detailed description of the surface organization of these pleomorphic virions. Our results, which further revealed a built-in mechanism controlling Gc membrane insertion for fusion, pave the way for immunogen design to protect against pathogenic hantaviruses.
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Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Orthohantavírus/química , Glicoproteínas/química , Glicoproteínas/ultraestrutura , Orthohantavírus/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Conformação Proteica , Vírus de RNA , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/ultraestrutura , Vírion , Internalização do VírusRESUMO
Andes virus (ANDV) is the only hantavirus transmitted between humans through close contact. We detected the genome and proteins of ANDV in breast milk cells from an infected mother in Chile who transmitted the virus to her child, suggesting gastrointestinal infection through breast milk as a route of ANDV person-to-person transmission.
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Infecções por Hantavirus , Orthohantavírus , Criança , Chile/epidemiologia , Feminino , Humanos , Transmissão Vertical de Doenças Infecciosas , Leite HumanoRESUMO
The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-ß, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Orthohantavírus/genética , Proteínas não Estruturais Virais/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Infecções por Hantavirus/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Interferon beta/genética , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/imunologia , Células Vero , Proteínas não Estruturais Virais/metabolismoRESUMO
The 2019 11th International Conference on Hantaviruses (ICH 2019) was organized by the International Society for Hantaviruses (ISH), and held on September 1-4, 2019, at the Irish College, in Leuven, Belgium. These ICHs have been held every three years since 1989. ICH 2019 was attended by 158 participants from 33 countries. The current report summarizes research presented on all aspects of hantavirology: ecology; pathogenesis and immune responses; virus phylogeny, replication and morphogenesis; epidemiology; vaccines, therapeutics and prevention; and clinical aspects and diagnosis.
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Orthohantavírus/patogenicidade , Pesquisa/tendências , Bélgica , Congressos como Assunto , Orthohantavírus/genética , Infecções por Hantavirus/epidemiologia , Infecções por Hantavirus/imunologia , Infecções por Hantavirus/terapia , HumanosRESUMO
Segmentation is one of the most important stages in the 3D reconstruction of macromolecule structures in cryo-electron microscopy. Due to the variability of macromolecules and the low signal-to-noise ratio of the structures present, there is no generally satisfactory solution to this process. This work proposes a new unsupervised particle picking and segmentation algorithm based on the composition of two well-known image filters: Anisotropic (Perona-Malik) diffusion and non-negative matrix factorization. This study focused on keyhole limpet hemocyanin (KLH) macromolecules which offer both a top view and a side view. Our proposal was able to detect both types of views and separate them automatically. In our experiments, we used 30 images from the KLH dataset of 680 positive classified regions. The true positive rate was 95.1% for top views and 77.8% for side views. The false negative rate was 14.3%. Although the false positive rate was high at 21.8%, it can be lowered with a supervised classification technique.
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Algoritmos , Hemocianinas/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Crioeletrônica , Bases de Dados Factuais , Substâncias MacromolecularesRESUMO
Phleboviruses constitute a large group of arthropod-borne viruses (arboviruses), mainly transmitted to their hosts by sandflies and ticks, occasionally by mosquitoes. These viruses have a worldwide distribution and many cause serious diseases - often fatal - in both domestic animals and humans. The global warming, the apparent wide distribution of arthropod reservoirs, and the increasing number of outbreaks show that phleboviruses must be taken seriously as emerging disease agents. This review proposes to focus on the early steps of phlebovirus infection, from virus binding to penetration into the cytosol. We address the most recent knowledge and advances in the entry of these viruses into vertebrate host cells, including virus receptors, cellular factors, endocytic pathways, and fusion.
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Arbovírus , Phlebovirus , Psychodidae , Carrapatos , Animais , Animais Domésticos , HumanosRESUMO
The hantavirus envelope glycoproteins Gn and Gc mediate virion assembly and cell entry, with Gc driving fusion of viral and endosomal membranes. Although the X-ray structures and overall arrangement of Gn and Gc on the hantavirus spikes are known, their detailed interactions are not. Here we show that the lateral contacts between spikes are mediated by the same 2-fold contacts observed in Gc crystals at neutral pH, allowing the engineering of disulfide bonds to cross-link spikes. Disrupting the observed dimer interface affects particle assembly and overall spike stability. We further show that the spikes display a temperature-dependent dynamic behavior at neutral pH, alternating between 'open' and 'closed' forms. We show that the open form exposes the Gc fusion loops but is off-pathway for productive Gc-induced membrane fusion and cell entry. These data also provide crucial new insights for the design of optimized Gn/Gc immunogens to elicit protective immune responses.
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Glicoproteínas/metabolismo , Orthohantavírus/metabolismo , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Internalização do Vírus , Sequência de Aminoácidos , Cristalografia por Raios X , Glicoproteínas/química , Glicoproteínas/genética , Orthohantavírus/genética , Orthohantavírus/fisiologia , Concentração de Íons de Hidrogênio , Fusão de Membrana , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.
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Vírus de Archaea/química , Proteínas de Fusão de Membrana/química , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Halorubrum/virologia , Fusão de Membrana , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/químicaRESUMO
The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.
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Caderinas/metabolismo , Orthohantavírus/fisiologia , Internalização do Vírus , Animais , Caderinas/química , Caderinas/deficiência , Caderinas/genética , Células Endoteliais/virologia , Feminino , Orthohantavírus/patogenicidade , Síndrome Pulmonar por Hantavirus/virologia , Haploidia , Interações Hospedeiro-Patógeno/genética , Humanos , Pulmão/citologia , Masculino , Mesocricetus/virologia , Domínios Proteicos , Protocaderinas , Vírus Sin Nombre/patogenicidade , Vírus Sin Nombre/fisiologiaRESUMO
Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens.
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Virus Puumala/química , Proteínas do Envelope Viral/química , Animais , Chlorocebus aethiops , Cristalografia por Raios X , Conformação Molecular , Conformação Proteica , Células VeroRESUMO
Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic ß-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single "fusion loop". We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal "tail" that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens.
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Orthobunyavirus/química , Orthohantavírus/química , Proteínas do Envelope Viral/química , Animais , Cristalografia , Glicoproteínas/química , Humanos , Conformação Proteica , Relação Estrutura-Atividade , Ressonância de Plasmônio de SuperfícieRESUMO
Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses.