scholarly journals Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons

2002 ◽  
Vol 76 (6) ◽  
pp. 3023-3030 ◽  
Author(s):  
Ralph S. Baric ◽  
Boyd Yount ◽  
Lisa Lindesmith ◽  
Patrick R. Harrington ◽  
Shermalyn R. Greene ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Norwalk Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Large Numbers ◽  
Virus Capsid Protein

ABSTRACT The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.

scholarly journals The SD1 Subdomain of Venezuelan Equine Encephalitis Virus Capsid Protein Plays a Critical Role in Nucleocapsid and Particle Assembly

2015 ◽  
Vol 90 (4) ◽  
pp. 2008-2020 ◽  
Author(s):  
Josephine M. Reynaud ◽  
Valeria Lulla ◽  
Dal Young Kim ◽  
Elena I. Frolova ◽  
Ilya Frolov
Keyword(s):  
Capsid Protein ◽  
Driving Forces ◽  
Critical Role ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Particle Assembly ◽  
Equine Encephalitis Virus ◽  
Amino Terminal ◽  
Virus Capsid Protein

ABSTRACTVenezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses.IMPORTANCEAlphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.

scholarly journals Venezuelan Equine Encephalitis Virus Capsid Protein Inhibits Nuclear Import in Mammalian but Not in Mosquito Cells

2008 ◽  
Vol 82 (8) ◽  
pp. 4028-4041 ◽  
Author(s):  
Svetlana Atasheva ◽  
Natalia Garmashova ◽  
Ilya Frolov ◽  
Elena Frolova
Keyword(s):  
Capsid Protein ◽  
Nuclear Import ◽  
Critical Role ◽  
The United States ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Small Proteins ◽  
Mosquito Cells

ABSTRACT Venezuelan equine encephalitis virus (VEEV) represents a continuous public health threat in the United States. It has the ability to cause fatal disease in humans and in horses and other domestic animals. We recently demonstrated that replicating VEEV interferes with cellular transcription and uses this phenomenon as a means of downregulating a cellular antiviral response. VEEV capsid protein was found to play a critical role in this process, and its ∼35-amino-acid-long peptide, fused with green fluorescent protein, functioned as efficiently as did the entire capsid. We detected a significant fraction of VEEV capsid associated with nuclear envelope, which suggested that this protein might regulate nucleocytoplasmic trafficking. In this study, we demonstrate that VEEV capsid and its N-terminal sequence efficiently inhibit multiple receptor-mediated nuclear import pathways but have no effect on the passive diffusion of small proteins. The capsid protein of the Old World alphavirus Sindbis virus and the VEEV capsid, with a previously defined frameshift mutation, were found to have no detectable effect on nuclear import. Importantly, the VEEV capsid did not noticeably interfere with nuclear import in mosquito cells, and this might play a critical role in the ability of the virus to develop a persistent, life-long infection in mosquito vectors. These findings demonstrate a new aspect of VEEV-host cell interactions, and the results of this study are likely applicable to other New World alphaviruses, such as eastern and western equine encephalitis viruses.

scholarly journals Nuclear Import and Export of Venezuelan Equine Encephalitis Virus Nonstructural Protein 2

2007 ◽  
Vol 81 (19) ◽  
pp. 10268-10279 ◽  
Author(s):  
Stephanie A. Montgomery ◽  
Robert E. Johnston
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nonstructural Protein ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Nonstructural Protein 2

ABSTRACT Many RNA viruses, which replicate predominantly in the cytoplasm, have nuclear components that contribute to their life cycle or pathogenesis. We investigated the intracellular localization of the multifunctional nonstructural protein 2 (nsP2) in mammalian cells infected with Venezuelan equine encephalitis virus (VEE), an important, naturally emerging zoonotic alphavirus. VEE nsP2 localizes to both the cytoplasm and the nucleus of mammalian cells in the context of infection and also when expressed alone. Through the analysis of a series of enhanced green fluorescent protein fusions, a segment of nsP2 that completely localizes to the nucleus of mammalian cells was identified. Within this region, mutation of the putative nuclear localization signal (NLS) PGKMV diminished, but did not obliterate, the ability of the protein to localize to the nucleus, suggesting that this sequence contributes to the nuclear localization of VEE nsP2. Furthermore, VEE nsP2 specifically interacted with the nuclear import protein karyopherin-α1 but not with karyopherin-α2, -3, or -4, suggesting that karyopherin-α1 transports nsP2 to the nucleus during infection. Additionally, a novel nuclear export signal (NES) was identified, which included residues L526 and L528 of VEE nsP2. Leptomycin B treatment resulted in nuclear accumulation of nsP2, demonstrating that nuclear export of nsP2 is mediated via the CRM1 nuclear export pathway. Disruption of either the NLS or the NES in nsP2 compromised essential viral functions. Taken together, these results establish the bidirectional transport of nsP2 across the nuclear membrane, suggesting that a critical function of nsP2 during infection involves its shuttling between the cytoplasm and the nucleus.

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