A plate-based high-throughput assay for virus stability and vaccine formulation

One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor ␣ 2 ␤ 1 integrin and showed high infectivity. A thermal assay further showed that the ␣ 2 ␤ 1 integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate. IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection.Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses. Picornaviruses are a large family of pathogens infecting humans and animals across the globe. The vast disease range caused by these viruses spans from simple rashes to paralysis and meningitis. It has been estimated that approximately half of the seasonal common-cold cases reported are caused by rhinoviruses, members of the family subgroup Enterovirus (1). Increased emphasis on enterovirus research is...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A Novel Open and Infectious Form of Echovirus 1

Myllynen

Kazmertsuk

Marjomäki

2016

“…The assay was run from 25°C to 95°C with readout every 0.33 s in an Mx3005P quantitative PCR (qPCR) instrument (Agilent Technologies). The ramp rate was 1°C/30 s. The Sybr Safe DNA gel stain dye was excited at 492 nm, and emission was read at 516 nm (56). The assay was also run at a constant temperature of 37°C for 12 h.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

Shakeel

Westerhuis

Ora

et al. 2015

Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCEHuman parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell. Human parechoviruses (HPeV) are single-stranded, positivesense RNA viruses in the Parechovirus genus within the Picornaviridae family (1). HPeV infections mainly cause mild gastrointestinal symptoms, although HPeV are also associated with more severe central nervous system symptoms, such as meningitis and neonatal sepsis (2-5). The HPeV genome is about 7,300 bases in length, enclose...

“…Heating enterovirus particles at 50 to 60°C for various amounts of time has been shown to be able to produce uncoating intermediates (135S) and result in genome ejecting, as well as producing empty (80S) particles indistinguishable from those observed in vivo (7,10,11). The Raman spectra of the particles that were heat treated at 50°C represent a state where the genome is partially ejected from the capsid, as confirmed by transmission electron microscopy (TEM) and thermal stability assays (29). We conclude that there are clear Raman markers which address the uncoating stage of the virion.…”

mentioning

confidence: 80%

Raman Spectroscopic Signatures of Echovirus 1 Uncoating

Ruokola

Dadu

Kazmertsuk

et al. 2014

In recent decades, Raman spectroscopy has entered the biological and medical fields. It enables nondestructive analysis of structural details at the molecular level and has been used to study viruses and their constituents. Here, we used Raman spectroscopy to study echovirus 1 (EV1), a small, nonenveloped human pathogen, in two different uncoating states induced by heat treatments. Raman signals of capsid proteins and RNA genome were observed from the intact virus, the uncoating intermediate, and disrupted virions. Transmission electron microscopy data revealed general structural changes between the studied particles. Compared to spectral characteristics of proteins in the intact virion, those of the proteins of the heat-treated particles indicated reduced ␣-helix content with respect to ␤-sheets and coil structures. Changes observed in tryptophan and tyrosine signals suggest an increasingly hydrophilic environment around these residues. RNA signals revealed a change in the environment of the genome and in its conformation. The ionized-carbonyl vibrations showed small changes between the intact virion and the uncoating intermediate, which points to cleavage of salt bridges in the protein structure during the uncoating process. In conclusion, our data reveal distinguishable Raman signatures of the intact, intermediate, and disrupted EV1 particles. These changes indicate structural, chemical, and solute-solvent alterations in the genome and in the capsid proteins and lay the essential groundwork for investigating the uncoating of EV1 and related viruses in real time. IMPORTANCEIn order to combat virus infection, we need to know the details of virus uncoating. We present here the novel Raman signatures for opened and intact echovirus 1. This gives hope that the signatures may be used in the near future to evaluate the ambient conditions in endosomes leading to virus uncoating using, e.g., coherent anti-Stokes Raman spectroscopy (CARS) imaging. These studies will complement structural studies on virus uncoating. In addition, Raman/CARS imaging offers the possibility of making dynamic live measurements in vitro and in cells which are impossible to measure by, for example, cryo-electron tomography. Furthermore, as viral Raman spectra can be overwhelmed with various contaminants, our study is highly relevant in demonstrating the importance of sample preparation for Raman spectroscopy in the field of virology. Picornaviruses are small, nonenveloped human pathogens able to cause a wide range of illnesses (1). The subgroup enteroviruses include clinically important echoviruses, coxsackieviruses, and poliovirus. These viruses cause a variety of diseases varying from mild infections to aseptic meningitis, heart muscle damage, and paralysis. Enteroviruses have also recently been associated with chronic diseases such as type 1 diabetes, cardiomyopathies, and atherosclerosis (2, 3). Structural details of these viruses have been obtained by means of X-ray crystallography or at a lower resolution by cryo-electron microsc...