Glycoprotein Interactions in the Assembly of Hantaviruses

Hantaviruses are one of the five genera of the vector-borne virus family Bunyaviridae. While other members of the family are transmitted via arthropods, hantaviruses are carried and transmitted by rodents and insectivores. Occasional transmission to humans occurs via inhalation of aerosolized rodent excreta. When transmitted to man hantaviruses cause hemorrhagic fever with renal syndrome (HFRS, in Eurasia, mortality ~10%) and hantavirus cardiopulmonary syndrome (HCPS, in the Americas, mortality ~40%). The single-stranded, negative-sense RNA genome of hantaviruses is in segments S, M and L that respectively encode for nucleocapsid (N), glycoproteins Gn and Gc, and RNA-dependent RNA-polymerase (RdRp or L protein). The genome segments, encapsidated by N protein to form ribonucleoprotein (RNP), are enclosed inside a lipid envelope decorated by spikes formed of Gn and Gc. The focus of this study was to understand the mechanisms and interactions through which the virion is formed and maintained. We observed that when extracted from virions both Gn and Gc favor homo- over hetero-oligomerization. The minimal glycoprotein complexes extracted from virion by detergent were observed, by using ultracentrifugation and gel filtration, to be tetrameric Gn and homodimeric Gc. These results led us to suggest a model where tetrameric Gn complexes are interconnected through homodimeric Gc units to form the grid-like surface architecture described for hantaviruses. This model was found to correlate with the three-dimensional (3D) reconstruction of virion surface created using cryo-electron tomography (cryo-ET). The 3D-density map showed the spike complex formed of Gn and Gc to be 10 nm high and to display a four-fold symmetry with dimensions of 15 nm times 15 nm. This unique square-shaped complex on a roughly round virion creates a hitch for the assembly, since a sphere cannot be broken into rectangles. Thus additional interactions are likely required for the virion assembly. In cryo-ET we observed that the RNP makes occasional contacts to the viral membrane, suggesting an interaction between the spike and RNP. We were able to demonstrate this interaction using various techniques, and showed that both Gn and Gc contribute to the interaction. This led us to suggest that in addition to the interactions between Gn and Gc, also the interaction between spike and RNP is required for assembly. We found galectin-3 binding protein (referred to as 90K) to co-purify with the virions and showed an interaction between 90K and the virion. Analysis of plasma samples taken from patients hospitalized for Puumala virus infection showed increased concentrations of 90K in the acute phase and the increased 90K level was found to correlate with several parameters that reflect the severity of acute HFRS. The results of these studies confirmed, but also challenged some of the dogmas on the structure and assembly of hantaviruses. We confirmed that Gn and RNP do interact, as long assumed. On the other hand we demonstrated that the glycoproteins Gn and Gc exist as homo-oligomers or appear in large hetero-oligomeric complexes, rather than form primarily heterodimers as was previously assumed. This work provided new insight into the structure and assembly of hantaviruses.Hantavirukset kuuluvat vektorivälitteisten virusten muodostamaan Bunyaviridae-perheeseen ja ovat yksi perheen viidestä suvusta. Muista perheeseen kuuluvista viruksista poiketen hantavirukset ovat jyrsijä- tai hyönteissyöjävälitteisiä. Ihmiseen hantavirus siirtyy hengitysilman kautta aerosolisoituneiden jyrsijöiden eritteiden mukana ja voi aiheuttaa joko munuaisoireisen verenvuotokuumeen tai sydän-keuhko-oireyhtymän. Hantaviruksen yksijuosteinen ja negatiivisäikeinen RNA-genomi on jakautunut kolmeen osaan. Genomin osat S, M ja L koodaavat neljää rakenteellista proteiinia: nukleokapsidiproteiini (N-proteiini), glykoproteiinit Gn ja Gc sekä viruksen polymeraasientsyymi. Viruksen genomin osat pakkautuvat N-proteiinin kanssa ribonukleoproteiiniksi (RNP), joka edelleen pakkautuu glykoproteiinien peittämän lipidivaipan sisään. Tämän väitöskirjatyön tarkoituksena oli oppia ymmärtämään viruspartikkelin muodostumiseen ja koossapitämiseen tarvittavia rakenneproteiinien välisiä vuorovaikutuksia. Huomasimme, että viruspartikkelista eristettäessä glykoproteiinit muodostavat pääasiassa homo-oligomeerisia yhdistelmiä. Määritimme viruksesta eristettyjen proteiiniyhdistelmien minimaalisiksi yksiköiksi yhdistelmän, jossa on neljä Gn-proteiinia ja yhdistelmän, jossa on kaksi Gc-proteiinia. Tämän perusteella loimme hypoteesin, jonka mukaan viruksen pinta muodostuu neljän Gn-proteiinin yhdistelmistä, jotka yhdistyvät toisiinsa kahden Gc-proteiinin kautta. Kryo-elektronitomografian avulla selvitetty viruksen pintarakenne vastasi luomaamme hypoteesia ja tämän rakennetutkimuksen mukaan glykoproteiinien muodostama piikki on 10 nm korkea ja nelisymmetrinen (15 nm x 15 nm). Havaitsemamme ainutlaatuinen nelisymmetrisyys luo ongelman viruksen pakkautumiselle, sillä pallon pintaa ei voi rikkoa neliöiksi. Tästä johtuen pakkautumiseen tarvittaneen Gn- ja Gc-proteiinien välisten vuorovaikutusten lisäksi myös muita vuorovaikutuksia. Rakennetutkimuksessa selvisi, että RNP on satunnaisesti kiinnittynyt glykoproteiinien muodostamiin piikkeihin. Osoitimme biokemiallisin tekniikoin tämän vuorovaikutuksen ja samalla kävi ilmi, että molemmat glykoproteiinit osallistuvat RNP:n ja piikin väliseen sitoutumiseen. Tuloksiemme perusteella virusten pakkautumiseen tarvitaan glykoproteiinien välisten vuorovaikutusten lisäksi piikin ja RNP:n välinen vuorovaikutus. Huomasimme, että viruksien mukana puhdistuksessa kulkeutuu tuntematon komponentti ja tunnistimme sen galectin-3:a sitovaksi proteiiniksi (90K), jonka osoitimme sitoutuvan viruspartikkeliin. Analysoimme Puumala-virusinfektion takia sairaalahoitoon joutuneiden myyräkuumepotilaiden plasmanäytteistä 90K-pitoisuuden ja huomasimme sen olevan koholla taudin akuutissa vaiheessa. Lisäksi havaitsimme kohonneen 90K-pitoisuuden korreloivan taudin vakavuutta kuvaavien kliinisten parametrien kanssa. Tämän väitöskirjatutkimuksen tulokset haastoivat, mutta toisaalta myös osoittivat toteen joitakin hantavirusten rakenteeseen ja pakkautumiseen liittyviä dogmia. Osoitimme, että Gn-proteiinin ja RNP:n välillä on vuorovaikutus, kuten aiemmin oli oletettu. Toisaalta osoitimme, että glykoproteiinit muodostavat mieluummin Gn-Gn ja Gc-Gc yhdistelmiä kuin Gn-Gc yhdistelmiä. Väitöskirjatyön lopputuloksena ymmärrämme nyt paremmin hantavirusten rakennetta ja pakkautumista

Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported “2× genome” and the “1.2× genome” infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions

Short ‘1.2× Genome’ Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2× genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported “2× genome” and the “1.2× genome” infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2× and 1.2× genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2× genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions

Short '1.2x Genome' Infectious Clone Initiates Kolmiovirid Replication in Boa constrictor Cells

Human hepatitis D virus (HDV) depends on hepatitis B virus co-infection and its glycoproteins for infectious particle formation. HDV was the sole known deltavirus for decades and believed to be a human-only pathogen. However, since 2018, several groups reported finding HDV-like agents from various hosts but without co-infecting hepadnaviruses. In vitro systems enabling helper virus-independent replication are key for studying the newly discovered deltaviruses. Others and we have successfully used constructs containing multimers of the deltavirus genome for the replication of various deltaviruses via transfection in cell culture. Here, we report the establishment of deltavirus infectious clones with 1.2x genome inserts bearing two copies of the genomic and antigenomic ribozymes. We used Swiss snake colony virus 1 as the model to compare the ability of the previously reported "2x genome" and the "1.2x genome" infectious clones to initiate replication in cell culture. Using immunofluorescence, qRT-PCR, immuno- and northern blotting, we found the 2x and 1.2x genome clones to similarly initiate deltavirus replication in vitro and both induced a persistent infection of snake cells. The 1.2x genome constructs enable easier introduction of modifications required for studying deltavirus replication and cellular interactions.Peer reviewe

LFRET, a novel rapid assay for anti-tissue transglutaminase antibody detection

The diagnosis of celiac disease (CD) is currently based on serology and intestinal biopsy, with detection of anti-tissue transglutaminase (tTG) IgA antibodies recommended as the first-line test. Emphasizing the increasing importance of serological testing, new guidelines and evidence suggest basing the diagnosis solely on serology without confirmatory biopsy. Enzyme immunoassays (EIAs) are the established approach for anti-tTG antibody detection, with the existing point-of-care (POC) tests lacking sensitivity and/or specificity. Improved POC methods could help reduce the underdiagnosis and diagnostic delay of CD. We have previously developed rapid homogenous immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET), and demonstrated their suitability in serodiagnostics with hanta- and Zika virus infections as models. In this study, we set out to establish a protein L -based TR-FRET assay (LFRET) for the detection of anti-tTG antibodies. We studied 74 patients with biopsy-confirmed CD and 70 healthy controls, with 1) the new tTG-LFRET assay, and for reference 2) a well-established EIA and 3) an existing commercial POC test. IgG depletion was employed to differentiate between anti-tTG IgA and IgG positivity. The sensitivity and specificity of the first-generation tTG-LFRET POC assay in detection of CD were 87.8% and 94.3%, respectively, in line with those of the reference POC test. The sensitivity and specificity of EIA were 95.9% and 91.9%, respectively. This study demonstrates the applicability of LFRET to serological diagnosis of autoimmune diseases in general and of CD in particular.Peer reviewe

Persistent Reptarenavirus and Hartmanivirus Infection in Cultured Boid Cells

Mammarenaviruses establish a persistent infection in their rodent and bat hosts, and the evidence suggests that reptarenaviruses and hartmaniviruses found in captive snakes act similarly. In snakes, reptarenaviruses cause boid inclusion body disease (BIBD), which is often associated with secondary infections. Snakes with BIBD usually carry more than a single pair of reptarenavirus S and L segments and occasionally demonstrate hartmanivirus coinfection. Here, we reported the generation of cell lines persistently infected with a single or two reptarenavirus(es) and a cell line with persistent reptarenavirus-hartmanivirus coinfection. By RT-PCR we demonstrated that the amount of viral RNA within the persistently infected cells remains at levels similar to those observed following initial infection. Using antibodies against the glycoproteins (GPs) and nucleoprotein (NP) of reptarenaviruses, we studied the levels of viral protein in cells passaged 10 times after the original inoculation and observed that the expression of GPs declines dramatically during persistent infection, unlike the expression of NP. Immunofluorescence (IF) staining served to demonstrate differences in the distribution of NP within the persistently infected compared to freshly infected cells. IF staining of cells inoculated with the viruses secreted from the persistently infected cell lines produced similar NP staining compared to cells infected with a traditionally passaged virus, suggesting that the altered NP expression pattern of persistently infected cells does not relate to changes in the virus. The cell cultures described herein can serve as tools for studying the coinfection and superinfection interplay between reptarenaviruses and studying the BIBD pathogenesis mechanisms. IMPORTANCE Mammarenaviruses cause a persistent infection in their natural rodent and bat hosts. Reptarenaviruses cause boid inclusion body disease (BIBD) in constrictor snakes, but it is unclear whether snakes are the natural host of these viruses. In this study, we showed that reptarenaviruses established a persistent infection in cultured Boa constrictor cells and that the persistently infected cells continued to produce infectious virus. Our results showed that persistent infection results from subsequent passaging of cells inoculated with a single reptarenavirus, two reptarenaviruses, or even when inoculating the cells with reptarenavirus and hartmanivirus (another arenavirus genus). The results further suggested that coinfection would not result in overt competition between the different reptarenaviruses, thus helping to explain the frequent reptarenavirus coinfections in snakes with BIBD. The established cell culture models of persistent infection could help to elucidate the role of coinfection and superinfection and potential immunosuppression as the pathogenic mechanisms behind BIBD

The fundamental role of endothelial cells in hantavirus pathogenesis

Peer reviewe

Persistent Reptarenavirus and Hartmanivirus Infection in Cultured Boid Cells

Mammarenaviruses cause a persistent infection in their natural rodent and bat hosts. Reptarenaviruses cause boid inclusion body disease (BIBD) in constrictor snakes, but it is unclear whether snakes are the natural host of these viruses. Mammarenaviruses establish a persistent infection in their rodent and bat hosts, and the evidence suggests that reptarenaviruses and hartmaniviruses found in captive snakes act similarly. In snakes, reptarenaviruses cause boid inclusion body disease (BIBD), which is often associated with secondary infections. Snakes with BIBD usually carry more than a single pair of reptarenavirus S and L segments and occasionally demonstrate hartmanivirus coinfection. Here, we reported the generation of cell lines persistently infected with a single or two reptarenavirus(es) and a cell line with persistent reptarenavirus-hartmanivirus coinfection. By RT-PCR we demonstrated that the amount of viral RNA within the persistently infected cells remains at levels similar to those observed following initial infection. Using antibodies against the glycoproteins (GPs) and nucleoprotein (NP) of reptarenaviruses, we studied the levels of viral protein in cells passaged 10 times after the original inoculation and observed that the expression of GPs declines dramatically during persistent infection, unlike the expression of NP. Immunofluorescence (IF) staining served to demonstrate differences in the distribution of NP within the persistently infected compared to freshly infected cells. IF staining of cells inoculated with the viruses secreted from the persistently infected cell lines produced similar NP staining compared to cells infected with a traditionally passaged virus, suggesting that the altered NP expression pattern of persistently infected cells does not relate to changes in the virus. The cell cultures described herein can serve as tools for studying the coinfection and superinfection interplay between reptarenaviruses and studying the BIBD pathogenesis mechanisms. IMPORTANCE Mammarenaviruses cause a persistent infection in their natural rodent and bat hosts. Reptarenaviruses cause boid inclusion body disease (BIBD) in constrictor snakes, but it is unclear whether snakes are the natural host of these viruses. In this study, we showed that reptarenaviruses established a persistent infection in cultured Boa constrictor cells and that the persistently infected cells continued to produce infectious virus. Our results showed that persistent infection results from subsequent passaging of cells inoculated with a single reptarenavirus, two reptarenaviruses, or even when inoculating the cells with reptarenavirus and hartmanivirus (another arenavirus genus). The results further suggested that coinfection would not result in overt competition between the different reptarenaviruses, thus helping to explain the frequent reptarenavirus coinfections in snakes with BIBD. The established cell culture models of persistent infection could help to elucidate the role of coinfection and superinfection and potential immunosuppression as the pathogenic mechanisms behind BIBD.Peer reviewe

Snake Deltavirus Utilizes Envelope Proteins of Different Viruses To Generate Infectious Particles

Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particlee03250-19s. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.Peer reviewe

Urine and Free Immunoglobulin Light Chains as Analytes for Serodiagnosis of Hantavirus Infection

Rapid point-of-care testing is a megatrend in infectious disease diagnosis. We have introduced a homogeneous immunoassay concept, which is based on the simultaneous binding of antigen and protein L to a given immunoglobulin molecule. The complex formation is detected utilizing time-resolved Förster resonance energy transfer between antigen-attached donor and acceptor-labeled protein L, hence the name LFRET. Here, we demonstrate that urine can be used as a sample matrix in LFRET-based serodiagnostics. We studied urine samples collected during the hospitalization and recovery of patients with acute Puumala orthohantavirus (PUUV) infection. We compared PUUV antibody-specific LFRET signals in urine to those in plasma, and found excellent correlation in the test outcomes The LFRET test from urine was positive in 40/40 patients with acute PUUV infection. PUUV causes a mild form of hemorrhagic fever with renal syndrome, characterized by acute kidney injury and proteinuria. Immunofluorescence and western blotting demonstrated PUUV-IgG and -IgA in urine, however, the presence of intact immunoglobulins did not fully explain the LFRET signals. We purified free light chains (FLCs) from both urine and serum of healthy volunteers and patients with acute PUUV infection, and verified the presence of antigen-specific FLCs. Antigen-specific FLCs provide a new means for non-invasive antibody detection and disease diagnosis