scholarly journals Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates

2020 ◽  
Vol 21 (20) ◽  
pp. 7686 ◽  
Author(s):  
Beáta Bozóki ◽  
János András Mótyán ◽  
Gyula Hoffka ◽  
David S. Waugh ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Protease Activity ◽  
Semliki Forest Virus ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Structural Basis ◽  
Structural Protein ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus

The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1′, and P2′ variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1′ amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.

scholarly journals Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses

2006 ◽  
Vol 80 (6) ◽  
pp. 2784-2796 ◽  
Author(s):  
Slobodan Paessler ◽  
Haolin Ni ◽  
Olga Petrakova ◽  
Rafik Z. Fayzulin ◽  
Nadezhda Yun ◽  
...  
Keyword(s):  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Challenge Virus ◽  
Equine Encephalitis Virus ◽  
Adult Mice ◽  
The Central Nervous System ◽  
Different Strains

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

scholarly journals Structure of Venezuelan equine encephalitis virus in complex with the LDLRAD3 receptor

Nature ◽  
2021 ◽  
Author(s):  
Katherine Basore ◽  
Hongming Ma ◽  
Natasha M. Kafai ◽  
Samantha Mackin ◽  
Arthur S. Kim ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Structural Basis ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Density Lipoprotein Receptor ◽  
Entry Receptor ◽  
Competition Assays

AbstractLDLRAD3 is a recently defined attachment and entry receptor for Venezuelan equine encephalitis virus (VEEV)1, a New World alphavirus that causes severe neurological disease in humans. Here we present near-atomic-resolution cryo-electron microscopy reconstructions of VEEV virus-like particles alone and in a complex with the ectodomains of LDLRAD3. Domain 1 of LDLRAD3 is a low-density lipoprotein receptor type-A module that binds to VEEV by wedging into a cleft created by two adjacent E2–E1 heterodimers in one trimeric spike, and engages domains A and B of E2 and the fusion loop in E1. Atomic modelling of this interface is supported by mutagenesis and anti-VEEV antibody binding competition assays. Notably, VEEV engages LDLRAD3 in a manner that is similar to the way that arthritogenic alphaviruses bind to the structurally unrelated MXRA8 receptor, but with a much smaller interface. These studies further elucidate the structural basis of alphavirus–receptor interactions, which could inform the development of therapies to mitigate infection and disease against multiple members of this family.

scholarly journals Inhibitors of Venezuelan Equine Encephalitis Virus Identified Based on Host Interaction Partners of Viral Non-Structural Protein 3

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1533
Author(s):  
Allison Bakovic ◽  
Nishank Bhalla ◽  
Farhang Alem ◽  
Catherine Campbell ◽  
Weidong Zhou ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Time Course ◽  
New World ◽  
Particle Production ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Structural Protein ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus ◽  
Host Interaction

Venezuelan equine encephalitis virus (VEEV) is a new world alphavirus and a category B select agent. Currently, no FDA-approved vaccines or therapeutics are available to treat VEEV exposure and resultant disease manifestations. The C-terminus of the VEEV non-structural protein 3 (nsP3) facilitates cell-specific and virus-specific host factor binding preferences among alphaviruses, thereby providing targets of interest when designing novel antiviral therapeutics. In this study, we utilized an overexpression construct encoding HA-tagged nsP3 to identify host proteins that interact with VEEV nsP3 by mass spectrometry. Bioinformatic analyses of the putative interactors identified 42 small molecules with the potential to inhibit the host interaction targets, and thus potentially inhibit VEEV. Three inhibitors, tomatidine, citalopram HBr, and Z-VEID-FMK, reduced replication of both the TC-83 strain and the Trinidad donkey (TrD) strain of VEEV by at least 10-fold in astrocytoma, astroglial, and microglial cells. Further, these inhibitors reduced replication of the related New World (NW) alphavirus Eastern equine encephalitis virus (EEEV) in multiple cell types, thus demonstrating broad-spectrum antiviral activity. Time-course assays revealed all three inhibitors reduced both infectious particle production and positive-sense RNA levels post-infection. Further evaluation of the putative host targets for the three inhibitors revealed an interaction of VEEV nsP3 with TFAP2A, but not eIF2S2. Mechanistic studies utilizing siRNA knockdowns demonstrated that eIF2S2, but not TFAP2A, supports both efficient TC-83 replication and genomic RNA synthesis, but not subgenomic RNA translation. Overall, this work reveals the composition of the VEEV nsP3 proteome and the potential to identify host-based, broad spectrum therapeutic approaches to treat new world alphavirus infections.

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