Molecular Organisation of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions

Molecular Organisation of Tick-Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions

Uukuniemi virus-like particles : a model system for bunyaviral assembly

Viruses are intracellular parasites that are unable to multiply except when inside a living cell of a host. Outside their host they remain inert. There are many different types of viruses that are classified into different virus families according to their size, nucleic acid composition (RNA or DNA), or the composition of their outer shell (naked or enveloped). Following entry, negative stranded viruses release their genome which is subsequently replicated, transcribed, and the different viral components are assembled into new virus particles, which are released from the host cell and ready to infect new cells. This thesis focuses on how the different viral parts assemble into an infectious particle inside the cell as well as on the identification of parts in the RNA genome important for viral replication. Uukuniemi virus (UUKV), a prototypic member of the Bunyaviridae family, has a segmented RNA genome with negative polarity. The three segments encode four structural proteins, two glycoproteins (GN and GC) located in the envelope, one nucleoprotein (N protein), and a RNA-dependent RNA polymerase (L protein). Flanking these open reading frames (ORFs) are non-coding regions (NCRs) containing cis-acting signals important for viral transcription, replication, encapsidation and packaging. These NCRs encompass one variable region, and one highly conserved region located in both the 5 and 3 terminal ends of the three segments, which are complementary to each other. To study the function of the NCRs we used a minigenome system developed for UUKV. In this system the viral protein coding sequence is replaced by sequences encoding a reporter protein. This minigenome is transfected into cells together with the N and L protein necessary for replication and transcription, after which reporter protein expression can be measured. In the first paper we studied the variable region of the NCRs and its effect on promoter strength and packaging efficiency. We performed this by comparing the activity of minigenomes that contained the NCRs derived from all three different segments. We found that the variable region is not only important for the regulation of promoter activity but also for packaging efficiency, since the three different minigenomes all showed different reporter activity. Next the assembly of the UUKV inside the cell was examined. In order to study packaging and assembly, we first developed a virus-like particle (VLP) system for UUKV. With the addition of the glycoprotein precursor encoding both glycoproteins GN and GC to the minigenome system, VLPs containing the minigenome are produced and released into the supernatant. These particles are able to infect new cells where reporter protein expression can be detected. Characterization of these VLPs revealed that they have similar size and surface morphology as wild-type UUKV. Moreover we demonstrated that only the two glycoproteins are required for generating VLPs, suggesting that UUKV budding is driven by the two glycoproteins. We further analyzed the importance of specific amino acids in the cytoplasmic tail of both glycoproteins, GN and GC, for packaging of the RNA genome into VLPs and the budding of UUKV. This was done by performing an alanine scan of the GN cytoplasmic tail (81 amino acids) and the GC cytoplasmic tail (5 amino acids), and analyzing the effect of these mutations on particle formation in our VLP system. We identified three regions in the GN tail (amino acids 21-25, 46-50 and 71-81) that are important for minigenome transfer of the VLPs. A more detailed analysis showed, four amino acids in the GN cytoplasmic tail involved in the packaging interaction with the ribonucleoproteins while two other amino acids are important for the budding into the Golgi compartment. Finally three amino acids in the GN and two in the GC cytoplasmic tail are important for the correct localization of the two glycoproteins in the cell. In conclusion, we show a mechanism of UUKV assembly and demonstrate the usefulness of our experimental system. We also postulate that the VLP system is a useful tool for analyzing packaging, assembly, and budding for other members of the Bunyavidae family

Tick-Borne Flaviviruses and the Type I Interferon Response

Flaviviruses are globally distributed pathogens causing millions of human infections every year. Flaviviruses are arthropod-borne viruses and are mainly transmitted by either ticks or mosquitoes. Mosquito-borne flaviviruses and their interactions with the innate immune response have been well-studied and reviewed extensively, thus this review will discuss tick-borne flaviviruses and their interactions with the host innate immune response

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs Is a weak interferon antagonist

Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans

Characterizing the cellular attachment receptor for Langat virus

Tick-borne encephalitis infections have increased the last 30 years. The mortality associated to this viral infection is 0.5 to 30% with a risk of permanent neurological sequelae, however, no therapeutic is currently available. The first steps of virus-cell interaction, such as attachment and entry, are of importance to understand pathogenesis and tropism. Several molecules have been shown to interact with tick-borne encephalitis virus (TBEV) at the plasma membrane surface, yet, no studies have proven that these are specific entry receptors. In this study, we set out to characterize the cellular attachment receptor(s) for TBEV using the naturally attenuated member of the TBEV complex, Langat virus (LGTV), as a model. Inhibiting or cleaving different molecules from the surface of A549 cells, combined with inhibition assays using peptide extracts from high LGTV binding cells, revealed that LGTV attachment to host cells is dependent on plasma membrane proteins, but not on glycans or glycolipids, and suggested that LGTV might use different cellular attachment factors on different cell types. Based on this, we developed a transcriptomic approach to generate a list of candidate attachment and entry receptors. Our findings shed light on the first step of the flavivirus life-cycle and provide candidate receptors that might serve as a starting point for future functional studies to identify the specific attachment and/or entry receptor for LGTV and TBEV

Complete Genome Sequence of a Low-Virulence Tick-Borne Encephalitis Virus Strain

We report here the complete genome sequence (GenBank accession no. KX268728) of tick-borne encephalitis strain HB171/11, isolated from an Ixodes ricinus tick from a natural focus where human neurological disease is rare. The strain shows unique characteristics in neuroinvasiveness and neurovirulence

Tick-Borne Encephalitis Virus Delays Interferon Induction and Hides Its Double-Stranded RNA in Intracellular Membrane Vesicles ▿

Tick-borne encephalitis virus (TBEV) (family Flaviviridae, genus Flavivirus) accounts for approximately 10,000 annual cases of severe encephalitis in Europe and Asia. Here, we investigated the induction of the antiviral type I interferons (IFNs) (alpha/beta IFN [IFN-α/β]) by TBEV. Using strains Neudörfl, Hypr, and Absettarov, we demonstrate that levels of IFN-β transcripts and viral RNA are strictly correlated. Moreover, IFN induction by TBEV was dependent on the transcription factor IFN regulatory factor 3 (IRF-3). However, even strain Hypr, which displayed the strongest IFN-inducing activity and the highest RNA levels, substantially delayed the activation of IRF-3. As a consequence, TBEV can keep the level of IFN transcripts below the threshold value that would permit the release of IFN by the cell. Only after 24 h of infection have cells accumulated sufficient IFN transcripts to produce detectable amounts of secreted IFNs. The delay in IFN induction appears not to be caused by a specific viral protein, since the individual expressions of TBEV C, E, NS2A, NS2B, NS3, NS4A, NS4B, NS5, and NS2B-NS3, as well as TBEV infection itself, had no apparent influence on specific IFN-β induction. We noted, however, that viral double-stranded RNA (dsRNA), an important trigger of the IFN response, is immunodetectable only inside intracellular membrane compartments. Nonetheless, the dependency of IFN induction on IFN promoter stimulator 1 (IPS-1) as well as the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) suggest the cytoplasmic exposure of some viral dsRNA late in infection. Using ultrathin-section electron microscopy, we demonstrate that, similar to other flaviviruses, TBEV rearranges intracellular membranes. Virus particles and membrane-connected vesicles (which most likely represent sites of virus RNA synthesis) were observed inside the endoplasmic reticulum. Thus, apparently, TBEV rearranges internal cell membranes to provide a compartment for its dsRNA, which is largely inaccessible for detection by cytoplasmic pathogen receptors. This delays the onset of IFN induction sufficiently to give progeny particle production a head start of approximately 24 h

Electron Cryo-Microscopy and Single-Particle Averaging of Rift Valley Fever Virus: Evidence for GN-GC Glycoprotein Heterodimers▿

Rift Valley fever virus (RVFV) is a member of the genus Phlebovirus within the family Bunyaviridae. It is a mosquito-borne zoonotic agent that can cause hemorrhagic fever in humans. The enveloped RVFV virions are known to be covered by capsomers of the glycoproteins GN and GC, organized on a T=12 icosahedral lattice. However, the structural units forming the RVFV capsomers have not been determined. Conflicting biochemical results for another phlebovirus (Uukuniemi virus) have indicated the existence of either GN and GC homodimers or GN-GC heterodimers in virions. Here, we have studied the structure of RVFV using electron cryo-microscopy combined with three-dimensional reconstruction and single-particle averaging. The reconstruction at 2.2-nm resolution revealed the organization of the glycoprotein shell, the lipid bilayer, and a layer of ribonucleoprotein (RNP). Five- and six-coordinated capsomers are formed by the same basic structural unit. Molecular-mass measurements suggest a GN-GC heterodimer as the most likely candidate for this structural unit. Both leaflets of the lipid bilayer were discernible, and the glycoprotein transmembrane densities were seen to modulate the curvature of the lipid bilayer. RNP densities were situated directly underneath the transmembrane densities, suggesting an interaction between the glycoprotein cytoplasmic tails and the RNPs. The success of the single-particle averaging approach taken in this study suggests that it is applicable in the study of other phleboviruses, as well, enabling higher-resolution description of these medically important pathogens