scholarly journals Novel Structural Features of Human Norovirus Capsid

2019 ◽  
Author(s):  
Jessica Devant ◽  
Götz Hofhaus ◽  
Grant S. Hansman
Keyword(s):  
Structural Changes ◽  
Expression System ◽  
Structural Diversity ◽  
Structural Features ◽  
Amino Acid Identity ◽  
Icosahedral Symmetry ◽  
Capsid Shell ◽  
Capsid Protein Vp1 ◽  
Cell Expression ◽  
Insect Cell Expression

ABSTRACTHuman noroviruses are a major cause of gastroenteritis, yet there are still no vaccines or antivirals available. Nevertheless, a number of vaccine candidates that are currently in clinical trials are composed of norovirus virus-like particles (VLPs). These VLPs are recognized as morphologically and antigenically similar to norovirus virions. An X-ray crystal structure of the prototype (GI.1) VLPs showed that the norovirus capsid has a T=3 icosahedral symmetry and is composed of 180 copies of the major capsid protein (VP1) that folds into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM structure of VLPs for two GII.4 noroviruses that were detected in 1974 and 2012. We showed that these VLPs had a T=4 symmetry and were composed of 240 copies of VP1. The VP1 on the T=4 VLPs adapted four quasi-equivalent subunits (termed A, B, C, and D), which formed two distinct dimers (A/B and C/D). We found that the T=4 protruding domain was elevated ~21 Å off the capsid shell, which was ~7 Å more than the previously determined for the T=3 GII.10 norovirus. Another interesting feature of the T=4 VLPs was a small cavity and flaplike structure located at the twofold axis. This structural feature was associated with the shell domain (D subunit) and disrupted the contiguous shell. Altogether, we showed that the T=4 VLPs had a number of structural similarities and differences with other noroviruses, but how these structural changes associate with norovirus virions could be important for vaccine studies.IMPORTANCEThe discovery that the GII.4 VLPs (identified in 1974 and 2012, termed CHDC-1974 and NSW-2012, respectively) have a T=4 symmetry is of major significance, since the NSW-2012 is clinically important and previous structural and biochemical studies assumed noroviruses have a T=3 symmetry and are composed of 180 copies of VP1. More importantly, NSW-2012 norovirus shared 96% amino acid identity with a GII.4 vaccine candidate and our data suggests that this vaccine might also have a T=4 symmetry. Although it is not clear if the T=4 VLPs were an artifact of the insect cell expression system, the T=4 VLP vaccines might not recognize equivalent epitopes on T=3 virions, which will be important for future neutralization studies. Finally, further studies with other norovirus genotypes and virions are clearly needed in order to determine the level of this structural diversity.

scholarly journals Heterologous Expression of Human Norovirus GII.4 VP1 Leads to Assembly of T=4 Virus-Like Particles

2019 ◽  
Author(s):  
Jessica Devant ◽  
Götz Hofhaus ◽  
David Bhella ◽  
Grant S. Hansman
Keyword(s):  
Structural Features ◽  
Amino Acid Identity ◽  
Icosahedral Symmetry ◽  
Binding Studies ◽  
Candidate Sequence ◽  
Virus Like Particles ◽  
Capsid Shell ◽  
Capsid Protein Vp1 ◽  
Norovirus Gii ◽  
D Subunit

ABSTRACTHuman noroviruses are a leading cause of acute gastroenteritis, yet there are still no vaccines or antivirals available. Expression of the norovirus capsid protein (VP1) in insect cells typically results in the formation of virus-like particles (VLPs) that are morphologically and antigenically comparable to native virions. Previous structural analysis of norovirus VLPs showed that the capsid has a T=3 icosahedral symmetry and is composed of 180 copies of VP1 that are folded into three quasi-equivalent subunits (A, B, and C). In this study, we determined the cryo-EM VLP structures of two GII.4 variants, termed CHDC-1974 and NSW-2012. Surprisingly, we found that greater than 95% of these GII.4 VLPs were larger than virions and 3D reconstruction showed that these VLPs exhibited T=4 icosahedral symmetry. We found that the T=4 VLPs showed several structural differences to the T=3 VLPs. The T=4 particles assemble from 240 copies of VP1 that adopt four quasi-equivalent conformations (A, B, C, and D) that form two distinct dimers, A/B and C/D. The T=4 protruding domains were elevated ∼21-Å off the capsid shell, which was ∼7-Å more than the previously studied GII.10 T=3 VLPs. A small cavity and flap-like structure at the icosahedral twofold axis disrupted the contiguous T=4 shell, a consequence of the D-subunit S-domains having smaller contact interfaces with neighboring dimers. Overall, our findings that old and new GII.4 VP1 sequences assemble T=4 VLPs might have implications for the design of potential future vaccines.IMPORTANCEThe discovery that the GII.4 VLPs have a T=4 symmetry is of significance, since this represents the first known T=4 calicivirus structure. Interestingly, the GII.4 2012 variant shares 96% amino acid identity with a current GII.4 VLP vaccine candidate sequence, which suggests that this vaccine might also have a T=4 symmetry. Our previous results with these GII.4 VLPs showed functional binding properties to antibodies and Nanobodies that were raised against T=3 (GII.10) VLPs. This suggests that the T=4 VLPs were antigenically comparable to T=3 particles, despite the obvious structural and size differences. On the other hand, these larger T=4 VLPs with novel structural features and possibly new epitopes might elicit antibodies that do not recognize equivalent epitopes on the T=3 VLPs. Further structural and binding studies using a library of GII.4-specific Nanobodies are planned in order to precisely investigate whether new epitopes are formed.

Protein production using the baculovirus-insect cell expression system

2013 ◽  
Vol 30 (1) ◽  
pp. 1-18 ◽  
Author(s):  
A. Contreras-Gómez ◽  
A. Sánchez-Mirón ◽  
F. García-Camacho ◽  
E. Molina-Grima ◽  
Y. Chisti
Keyword(s):  
Insect Cell ◽  
Protein Production ◽  
Expression System ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

Activation of recombinant trp by thapsigargin in Sf9 insect cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1501-C1505 ◽  
Author(s):  
L. Vaca ◽  
W. G. Sinkins ◽  
Y. Hu ◽  
D. L. Kunze ◽  
W. P. Schilling
Keyword(s):  
Expression System ◽  
Cation Selectivity ◽  
Drosophila Protein ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Mammalian Protein ◽  
Entry Mechanism ◽  
Proline Rich Region ◽  
Cell Expression System

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

scholarly journals Calicivirus VP2 forms a portal to mediate endosome escape

2018 ◽  
Author(s):  
Michaela Conley ◽  
Marion McElwee ◽  
Liyana Azmi ◽  
Mads Gabrielsen ◽  
Olwyn Byron ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Conformational Changes ◽  
Structural Changes ◽  
Host Cells ◽  
Major Step ◽  
Endosomal Membrane ◽  
Endosome Escape ◽  
Animal Pathogens ◽  
Capsid Shell ◽  
Capsid Protein Vp1

AbstractTo initiate the infectious process, many viruses enter their host cells by triggering endocytosis following receptor engagement. The mechanism by which non-enveloped viruses, such as the caliciviruses, escape the endosome is however poorly understood. TheCaliciviridaeinclude many important human and animal pathogens, most notably norovirus, the cause of winter vomiting disease. Here we show that VP2, a minor capsid protein encoded by all caliciviruses, forms a large portal assembly at a unique three-fold symmetry axis following receptor engagement. This feature surrounds an open pore in the capsid shell. We hypothesise that the VP2 portal complex is the means by which the virus escapes the endosome, pene-trating the endosomal membrane to release the viral genome into the cytoplasm. Cryogenic electron microscopy (cryoEM) and asymmetric reconstruction were used to investigate structural changes in the capsid of feline calicivirus (FCV) that occur when the virus binds to its cellular receptor junctional adhesion molecule-A (fJAM-A). Near atomic-resolution structures were calculated for the native virion alone and decorated with soluble receptor fragments. We present atomic models of the major capsid protein VP1 in the presence and absence of fJAM-A, revealing the contact interface and conformational changes brought about by the interaction. Furthermore, we have calculated an atomic model of the portal protein VP2 and revealed the structural changes in VP1 that lead to pore formation. While VP2 was known to be critical for the production of infectious virus, its function has been hitherto undetermined. Our finding that VP2 assembles a portal that is likely responsible for endosome escape represents a major step forward in our understanding of both theCaliciviridaeand icosahedral RNA containing viruses in general.

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