Progress 08/15/06 to 08/14/09
Outputs
OUTPUTS: Aim 1: Cryo-EM and tomographic reconstruction of PRRSV virions have revealed the 3D structure of the PRRSV virion in their native state. Aim 2: Progress in this aim was described in the report for 2008. Briefly, PRRSV proteins NSP1a, NSP1b, NSP2 (N-terminal domain), M and GP5 endodomains and GP2 ectodomain were expressed in E. coli; NSP1a, NSP1b and NSP2 also in Pichia, and GP2 in Drosophila. Most proteins were insoluble or yields too low for crystallization. M was purified and subjected to NMR and CD analysis, showing that the protein is disordered and flexible in solution. In vitro binding assays of M and N were conducted, and did not find any significant interaction between the two proteins. In the current reporting period, the project work has focused on Aim 1. The results from this study were presented annual PRRS symposium in Chicago, Dec 2008. The results were also presented at an invited lecture at the University of Nebraska, Lincoln, in March 2009. The tomographic reconstruction results were published in J. Gen. Virol. in March 2009. PARTICIPANTS: Individuals: Terje Dokland, Principal Investigator, has been responsible for the overall design and concept of the project. The PI has also been directly carrying out cryo-EM, tomographic data collection and reconstruction, prepared the manuscript and presented the work at meetings. Cindy Rodenburg, research assistant, has done expression and purification of M, GP5, GP2 and non-structural proteins in E. coli and Drosophila systems and also assisted with the electron microscopy and preparation of viruses for microscopy. Michael Spilman, graduate student, has worked on the cryo-EM and tomographic data collection and reconstruction (aim 1). Partner organizations: South Dakota State University, Brookings, SD. Collaborators: Eric Nelson, Dept. of Veterinary Science, South Dakota State University, has supplied all the purified virus used for the EM study, as well as antibodies. Craig Welbon, Dept. of Veterinary Science, South Dakota State University, grew and purified the virus samples that were used. Ying Fang, Dept. of Veterinary Science, South Dakota State University, has supplied clones of the non-structural proteins to be used for expression. Dongwan Yoo, University of Illinois, Champagne-Urbana, has supplied clones of structural proteins. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: outlined in Aim 1 of the original proposal. The scope of this aim 1 thus went beyond what was originally proposed. The crystallographic part of the project (Aim 2) was assigned a lower priority, since preliminary studies identified several technical difficulties, and the small amount of funding available did not allow any full-time personnel to be allocated to the project. An additional extension to the project has been to explore antibody labeling of virus followed by negative stain or cryo-EM imaging to localize specific proteins and epitopes on the viral surface. Several antibodies were tested, but none were found to be suitable for this purpose. We are currently exploring this avenue with Dr. Fernando Osorio at the University of Nebraska, and hope to obtain better, monospecific monoclonal antibodies that recognize external, accessible epitopes on the viral surface.
Impacts
The cryo-EM and tomographic structures have revealed that PRRSV virions have a pleiomorphic morphology with a smooth outer surface and a hollow, double-layered core. The structures have suggested that the core is organized as a double-layered ribbon consisting of N dimers and RNA bundled into a hollow ball. These results have challenged the existing paradigm for arterivirus structure, based on traditional EM studies, which posited that PRRSV and other arteriviruses has an isometric, icosahedral core. It also suggests a more fundamental structural relationship between the coronaviruses and arteriviruses that reflects their already established genomic similarities, with implications for understanding viral evolutionary relationships. These novel results were received with great enthusiasm at the PRRS meeting in Chicago, demonstrating that such structural studies fill a void in our understanding of arterivirus structure, which may eventually also have clinical and therapeutic implications.
Publications
- M.S. Spilman, C. Welbon, E. Nelson, T. Dokland. 2009. Cryo-electron tomography of porcine reproductive and respiratory syndrome virus: organization of the nucleocapsid. J. Gen. Virol. 90, 527-535.
- Dokland, T., Spilman, M.S., Welbon, C., Nelson, E. 2008. Cryo-electron tomography of PRRSV. Proceedings of the 2008 International PRRS Symposium, Dec 5-6, Chicago, IL, pg. 24.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: Aim 1: PRRSV virions were grown in MARC-145 cells, imaged by cryo-EM and subjected to tomographic reconstruction procedures, resulting in a 3D structure of the virion. Aim 2: PRRSV proteins NSP1a, NSP1b, NSP2 (N-terminal domain), M and GP5 endodomains and GP2 ectodomain were expressed in E. coli; NSP1a, NSP1b and NSP2 also in Pichia, and GP2 in Drosophila. Most proteins were insoluble or yields too low for crystallization. M was purified and subjected to NMR and CD analysis, showing that the protein is disordered and flexible in solution. In vitro binding assays of M and N were conducted, and did not find any significant interaction between the two proteins. In the reporting period, two graduate students and one undergraduate has been involved in the work. The results have been presented at two meetings: the annual PRRS symposium in Chicago, Dec 2007, and the FASEB conference on virus structure and assembly, Saxtons River, VT, June 2008. A publication describing the cryo-tomographic results was prepared and submitted to J. Gen. Virol., and is currently under review. PARTICIPANTS: Individuals: Terje Dokland, Principal Investigator, has been responsible for the overall design and concept of the project. The PI has also been directly carrying out cryo-EM, tomographic data collection and reconstruction, prepared the manuscript and presented the work at meetings. Cindy Rodenburg, research assistant, has done expression and purification of M, GP5, GP2 and non-structural proteins in E. coli and Drosophila systems (aim 2). Michael Spilman, graduate student, has worked on the cryo-EM and tomographic data collection and reconstruction (aim 1). Partner organizations: South Dakota State University, Brookings, SD. Collaborators: Eric Nelson, Dept. of Veterinary Science, South Dakota State University, has supplied all the purified virus used for the EM study (Aim 1). Ying Fang, Dept. of Veterinary Science, South Dakota State University, has supplied clones of the non-structural proteins to be used for expression. Dongwan Yoo, University of Illinois, Champagne-Urbana, has supplied clones of structural proteins. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: The main thrust of the project has shifted to the cryo-EM and tomographic structure determination, which has gone beyond what was originally presented in Aim 1 of the proposal. The crystallographic part of the project (Aim 2) has been assigned a lower priority, since preliminary studies identified several technical difficulties, and the small amount of funding available has not allowed any full-time personnel to be allocated to the project. Work continues on this aim, however, and focuses on the non-structural proteins NSP1a, NSP1b and NSP7.
Impacts
Aim1: The cryo-EM and tomographic structures have revealed the pleiomorphic morphology of PRRSV, the lipid bilayer and smooth outer surface as well as a hollow, double-layered core. The structures have suggested that the core is organized as a double-layered ribbon consisting of N dimers and RNA bundled into a hollow ball. The organization is expected to be similar to that of coronaviruses. Aim 2: Aim 2 has been difficult to complete, in part due to the resistance of most of the PRRSV proteins to E. coli expression, purification and crystallization, and in part due to insufficient funding for personnel and supplies to proceed with eukaryotic expression systems. The lack of interaction between N and M in vitro is consistent with the proposed model for core structure. Current knowledge of the structures of PRRSV and its proteins is very limited. Thus, structural information, such as that provided by cryo-electron microscopy and X-ray crystallography will be highly advantageous to understand the pathogenicity and immunogenicity of the virus. The cryo-tomographic reconstruction provides a baseline for understanding the organization and assembly of the virus, which is ultimately linked to its pathogenic life cycle and could be used to direct preventive approaches against the virus.
Publications
- M.S. Spilman, C. Welbon, E. Nelson, T. Dokland (2008) Cryo-electron tomography of porcine reproductive and respiratory syndrome virus (PRRSV): organization of the nucleocapsid. Submitted to J. Gen. Virol.
- Dokland, T., Deshpande, A., Fang, Y., Nelson, E. (2007). Cryo-electron microscopy of PRRSV virions. (Annual PRRS Symposium, Chicago, IL)
- Dokland, T., Spilman, M.S., Welbon, C., Nelson, E. (2008) Cryo-electron tomography and tomographic reconstruction of PRRSV. (FASEB Conference on Virus Structure and Assembly, Saxtons River, VT, June 2008)
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Progress 08/15/06 to 08/14/07
Outputs
The overall aim of this study is to provide structural information on porcine reproductive and respiratory syndrome virus (PRRSV) and viral proteins through electron microscopy and X-ray crystallographic analysis. Progress has been made in the two aims of the project. AIM 1--Image PRRSV virions by cryo-EM: PRRSV is difficult to grow to the high titers required for structural studies. With our collaborator, Dr. Eric Nelson at SDSU, we have optimized growth conditions of the field isolate SD-23983 to a titer of 10e7 pfu/ml in cell supernatants with minimal cell lysis. These culture supernatants were purified and concentrated to about 10e9 pfu/ml by centrifugation and prepared for cryo-EM by rapid freezing in liquid ethane. Images were collected on a 4k x 4k pixel CCD detector at a magnification of 65,000x. The virions appear as round or egg-shaped particles with an average diameter of 58 nm. The lipid bilayer of the envelope is clearly visible, and the particles display
a smooth outer surface with only a few protruding features, presumably the envelope proteins. The virions contain an internal, hollow core of 39 nm average diameter, which is separated from the envelope by a 2-3 nm gap. Since the virus particles are individually different, like those of e.g. influenza or HIV, reconstruction techniques employing averaging procedures are not available. We plan to use tomographic reconstruction to determine the three-dimensional structure of individual PRRSV virions. This is the first time an arterivirus has been visualized in this detail, providing much insight into virus infection, immunity and assembly. AIM 2--Determine structures of PRRSV proteins: We have overexpressed in E. coli and purified mg quantities of several PRRSV proteins, including full-length and truncated N (nucleocapsid), endodomains of M and GP5, GP2 ectodomain (membrane proteins) and full-length and truncated NSP1alpha, NSP1beta and NSP2 (non-structural proteins). In a comparative
study, we have also expressed and purified N from the related virus EAV. PRRSV and EAV N domains have been crystallized and the structures were solved crystallographically. Full-length N has been used to produce capsid-like structures that were subjected to cryo-EM, revealing a heterogeneous collection of shapes and sizes that appear to reflect the structure of the nucleocapsid in the virions (Aim 1). The M endodomain is highly expressed in a soluble form, but crystallization has so far not been feasible, probably due to high flexibility in solution. We are instead considering using NMR to study the structure of this protein. Most of the other proteins are insoluble upon overexpression in E. coli. The current focus is on NSP2 expressed at low temperature with slow induction in E. coli, and GP2, which is being expressed in a Drosophila system. High resolution structures would be highly desirable to understand roles of the various membrane proteins and non-structural proteins in PRRSV
infection and immunity.
Impacts
Outbreaks of PRRSV wreak havoc on pig farms worldwide and efforts to control this important disease have not been successful. The mechanisms of PRRSV infection and generation of immunity are currently poorly understood. Structural information on PRRSV and the viral proteins such as provided by cryo-electron microscopy and X-ray crystallography will be highly advantageous to understand these mechanisms. Such knowledge would be invaluable in directing preventive approaches against this virus.
Publications
- Deshpande, A., Wang, S., Walsh, M. and Dokland, T. 2007. Structure of the equine arterivirus nucleocapsid protein reveals a dimer-dimer arrangement. Acta Cryst. D 63, 581-586.
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