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.

scholarly journals Application of In Silico and HTS Approaches to Identify Nuclear Import Inhibitors for Venezuelan Equine Encephalitis Virus Capsid Protein: A Case Study

2020 ◽  
Vol 8 ◽  
Author(s):  
Sharon Shechter ◽  
David R. Thomas ◽  
David A. Jans
Keyword(s):  
Drug Design ◽  
Nuclear Import ◽  
In Silico ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Nuclear Targeting ◽  
Equine Encephalitis Virus ◽  
Screening Approaches

The development of new drugs is costly and time-consuming, with estimates of over $US1 billion and 15 years for a product to reach the market. As understanding of the molecular basis of disease improves, various approaches have been used to target specific molecular interactions in the search for effective drugs. These include high-throughput screening (HTS) for novel drug identification and computer-aided drug design (CADD) to assess the properties of putative drugs before experimental work begins. We have applied conventional HTS and CADD approaches to the problem of identifying antiviral compounds to limit infection by Venezuelan equine encephalitis virus (VEEV). Nuclear targeting of the VEEV capsid (CP) protein through interaction with the host nuclear import machinery has been shown to be essential for viral pathogenicity, with viruses incapable of this interaction being greatly attenuated. Our previous conventional HTS and in silico structure-based drug design (SBDD) screens were successful in identifying novel inhibitors of CP interaction with the host nuclear import machinery, thus providing a unique opportunity to assess the relative value of the two screening approaches directly. This focused review compares and contrasts the two screening approaches, together with the properties of the inhibitors identified, as a case study for parallel use of the two approaches to identify antivirals. The utility of SBDD screens, especially when used in parallel with traditional HTS, in identifying agents of interest to target the host–pathogen interface is highlighted.

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 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 mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research

2015 ◽  
Vol 89 (16) ◽  
pp. 8292-8303 ◽  
Author(s):  
Changqing Li ◽  
Jaime Guillén ◽  
Nadia Rabah ◽  
Alexandre Blanjoie ◽  
Françoise Debart ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Health Concern ◽  
Viral Mrna ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Mrna Capping ◽  
First Time

ABSTRACTAlphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group fromS-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m7GMP-nsP1 adduct. Subsequent transfer of m7GMP onto the 5′ end of the viral mRNA has not been demonstratedin vitroyet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m7G-cap monoclonal antibody, respectively. The GT reaction is stimulated byS-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m7GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5′-diphosphate RNA oligonucleotide whose sequence corresponds to the 5′ end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research.IMPORTANCEEmergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5′ diphosphate synthetic RNA mimicking the 5′ end of an alphavirus mRNA was observedin vitrofor the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.

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