Cryo-Electron Microscopy of Aura Viruses

1998 ◽  
Vol 4 (S2) ◽  
pp. 946-947
Author(s):  
W. Zhang ◽  
N. H. Olson ◽  
B. R. McKinney ◽  
R. J. Kuhn ◽  
T. S. Baker
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Single Copy ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Subgenomic Rna ◽  
Enveloped Viruses ◽  
Virus Particles ◽  
Cryo Electron Microscopy

Alphaviruses are a group of enveloped viruses in the Togaviridae family. Studies of several alphaviruses, including Ross River, Sindbis and Semliki Forest viruses, by cryo-electron microscopy (cryo-EM), three-dimensional (3D) image resconstruction and other techniques have illustrated that these spherical viruses have a T=4, multi-layered structure.Aura virus, which is closely related to Sindbis, was first isolated in South America. Unlike the other alphaviruses, both genomic RNA (12kb, 49S) and subgenomic RNA(4.2kb, 26S) are encapsidated efficiently and form mature virions. Studies on negatively-stained virus particles demonstrated that there are two major size classes. The first contains particles of ∼72nm diameter, which are most similar to wild type virus, whereas the second class includes particles of ∼62nm in diameter. The 72nm particles are believed to have one copy of genomic RNA or one to three copies of subgenomic RNA, and a T=4 structure. The 62nm particles probably only have a single copy of subgenomic RNA and are presumed to be T=3 structures.

scholarly journals Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

2013 ◽  
Vol 20 (1) ◽  
pp. 164-174 ◽  
Author(s):  
Gabriella Kiss ◽  
Xuemin Chen ◽  
Melinda A. Brindley ◽  
Patricia Campbell ◽  
Claudio L. Afonso ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Influenza A ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Nitrilotriacetic Acid ◽  
Dimensional Structure ◽  
Enveloped Viruses ◽  
Cryo Electron Microscopy ◽  
Human Immunodeficiency Virus Virus

AbstractElectron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

scholarly journals Cryo-electron microscopy and three-dimensional reconstructions of hepatitis C virus particles

Virology ◽  
2007 ◽  
Vol 367 (1) ◽  
pp. 126-134 ◽  
Author(s):  
Xuekui Yu ◽  
Ming Qiao ◽  
Ivo Atanasov ◽  
Zongyi Hu ◽  
Takanobu Kato ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Three Dimensional ◽  
Virus Particles ◽  
Cryo Electron Microscopy

scholarly journals The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Tomáš Kouba ◽  
Jiří Pospíšil ◽  
Jarmila Hnilicová ◽  
Hana Šanderová ◽  
Ivan Barvík ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Polymerase ◽  
Conformational Changes ◽  
Mycobacterium Smegmatis ◽  
Drug Target ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Cryo Electron Microscopy ◽  
Bacterial Rna ◽  
High Degree

ABSTRACT Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP. IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

scholarly journals Modification of the 5′ Terminus of Sindbis Virus Genomic RNA Allows nsP4 RNA Polymerases with Nonaromatic Amino Acids at the N Terminus To Function in RNA Replication

2003 ◽  
Vol 77 (4) ◽  
pp. 2301-2309 ◽  
Author(s):  
Yukio Shirako ◽  
Ellen G. Strauss ◽  
James H. Strauss
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Sindbis Virus ◽  
Rna Synthesis ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Minus Strand ◽  
Genomic Rna ◽  
Wild Type ◽  
N Terminus

ABSTRACT We have previously shown that Sindbis virus RNA polymerase requires an N-terminal aromatic amino acid or histidine for wild-type or pseudo-wild-type function; mutant viruses with a nonaromatic amino acid at the N terminus of the polymerase, but which are otherwise wild type, are unable to produce progeny viruses and will not form a plaque at any temperature tested. We now show that such mutant polymerases can function to produce progeny virus sufficient to form plaques at both 30 and 40°C upon addition of AU, AUA, or AUU to the 5′ terminus of the genomic RNA or upon substitution of A for U as the third nucleotide of the genome. These results are consistent with the hypothesis that (i) 3′-UA-5′ is required at the 3′ terminus of the minus-strand RNA for initiation of plus-strand genomic RNA synthesis; (ii) in the wild-type virus this sequence is present in a secondary structure that can be opened by the wild-type polymerase but not by the mutant polymerase; (iii) the addition of AU, AUA, or AUU to the 5′ end of the genomic RNA provides unpaired 3′-UA-5′ at the 3′ end of the minus strand that can be utilized by the mutant polymerase, and similarly, the effect of the U3A mutation is to destabilize the secondary structure, freeing 3′-terminal UA; and (iv) the N terminus of nsP4 may directly interact with the 3′ terminus of the minus-strand RNA for the initiation of the plus-strand genomic RNA synthesis. This hypothesis is discussed in light of our present results as well as of previous studies of alphavirus RNAs, including defective interfering RNAs.

scholarly journals Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

2016 ◽  
Vol 90 (19) ◽  
pp. 8542-8551 ◽  
Author(s):  
Lauren M. Drouin ◽  
Bridget Lins ◽  
Maria Janssen ◽  
Antonette Bennett ◽  
Paul Chipman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Intramolecular Interactions ◽  
Single Amino Acid ◽  
Fold Axis ◽  
Wild Type ◽  
Scanning Calorimetry ◽  
Genome Packaging ◽  
Cryo Electron Microscopy ◽  
Empty Capsid ◽  
Packaging Efficiency

ABSTRACTThe adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

scholarly journals Cryomesh™: A New Substrate for Cryo-Electron Microscopy

2010 ◽  
Vol 16 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Craig Yoshioka ◽  
Bridget Carragher ◽  
Clinton S. Potter
Keyword(s):  
Electron Microscopy ◽  
Semiconductor Industry ◽  
Three Dimensional ◽  
Quality Of Data ◽  
Temperature Conductivity ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Using Data ◽  
Collection Methods

AbstractHere we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

scholarly journals The Three-dimensional Constitution of Micelle Forming Surfactants as Studied by Cryo Electron Microscopy and Tomography

2006 ◽  
Vol 12 (S02) ◽  
pp. 1544-1545
Author(s):  
N Chebotareva ◽  
PH H Bomans ◽  
F De Haas ◽  
D Hubert ◽  
P Frederik ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Three Dimensional ◽  
Cryo Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2006 in Chicago, Illinois, USA, July 30 – August 3, 2006

scholarly journals Inactivation of hantaviruses by N-ethylmaleimide preserves virion integrity

2011 ◽  
Vol 92 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Tomas Strandin ◽  
Jussi Hepojoki ◽  
Hao Wang ◽  
Antti Vaheri ◽  
Hilkka Lankinen
Keyword(s):  
Cell Attachment ◽  
Buoyant Density ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Thiol Groups ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Nitrobenzoic Acid ◽  
The Family

Thiol groups of cysteine residues are crucial for the infectivity of various enveloped viruses, but their role in the infectivity of viruses of the family Bunyaviridae has thus far not been studied. This report shows that thiol groups are essential to the infectivity of hantaviruses. Alkylation of the thiol functional groups using the membrane-permeable compound N-ethylmaleimide (NEM) and membrane-impermeable compound 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) showed NEM to be a highly effective inactivator of Puumala and Tula hantaviruses. The NEM-inactivated hantavirus maintained the buoyant density of the wild-type virus. Furthermore, the antigenicity of glycoproteins and the cell attachment capacity of virions were retained at NEM concentrations that totally abolished virus infectivity. These results signified preservation of virion integrity following inactivation with NEM, making chemically inactivated virions valuable research antigens. It was demonstrated with biotin-conjugated maleimide, a mechanistic analogue of NEM, that all the structural proteins of hantavirus were sensitive towards thiol alkylation. In contrast to hantaviruses, NEM did not abolish Uukuniemi phlebovirus infectivity to the same extent. This indicates differences in the use of free thiols in virus entry among members of the family Bunyaviridae.

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