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

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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Host translation shutoff mediated by non-structural protein 2 is a critical factor in the antiviral state resistance of Venezuelan equine encephalitis virus

Virology ◽

10.1016/j.virol.2016.06.005 ◽

2016 ◽

Vol 496 ◽

pp. 147-165 ◽

Cited By ~ 20

Author(s):

Nishank Bhalla ◽

Chengqun Sun ◽

L.K. Metthew Lam ◽

Christina L. Gardner ◽

Kate D. Ryman ◽

...

Keyword(s):

Critical Factor ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Structural Protein ◽

Venezuelan Equine Encephalitis ◽

Antiviral State ◽

Equine Encephalitis Virus ◽

State Resistance

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Venezuelan Equine Encephalitis Virus Variants Lacking Transcription Inhibitory Functions Demonstrate Highly Attenuated Phenotype

Journal of Virology ◽

10.1128/jvi.02252-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 71-82 ◽

Cited By ~ 25

Author(s):

Svetlana Atasheva ◽

Dal Young Kim ◽

Elena I. Frolova ◽

Ilya Frolov

Keyword(s):

Public Health ◽

New World ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Venezuelan Equine Encephalitis ◽

Subsequent Infection ◽

Vaccine Candidates ◽

Health Threat ◽

Equine Encephalitis Virus ◽

Public Health Threat

ABSTRACTAlphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses.IMPORTANCEVenezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.

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Venezuelan Equine Encephalitis Virus nsP3 Phosphorylation Can Be Mediated by IKKβ Kinase Activity and Abrogation of Phosphorylation Inhibits Negative-Strand Synthesis

Viruses ◽

10.3390/v12091021 ◽

2020 ◽

Vol 12 (9) ◽

pp. 1021

Author(s):

Allison Bakovic ◽

Nishank Bhalla ◽

Stephanie Kortchak ◽

Chengqun Sun ◽

Weidong Zhou ◽

...

Keyword(s):

Mass Spectrometry ◽

Viral Replication ◽

Particle Production ◽

Kinase Activity ◽

Venezuelan Equine Encephalitis Virus ◽

Amino Acid Sequences ◽

Encephalitis Virus ◽

Venezuelan Equine Encephalitis ◽

Negative Strand ◽

Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV), a mosquito transmitted alphavirus of the Togaviridae family, can cause a highly inflammatory and encephalitic disease upon infection. Although a category B select agent, no FDA-approved vaccines or therapeutics against VEEV currently exist. We previously demonstrated NF-κB activation and macromolecular reorganization of the IKK complex upon VEEV infection in vitro, with IKKβ inhibition reducing viral replication. Mass spectrometry and confocal microscopy revealed an interaction between IKKβ and VEEV non-structural protein 3 (nsP3). Here, using western blotting, a cell-free kinase activity assay, and mass spectrometry, we demonstrate that IKKβ kinase activity can directly phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5. Alanine substitution mutations at sites 204/5, 142, or 134/5 reduced VEEV replication by >30-100,000-fold corresponding to a severe decrease in negative-strand synthesis. Serial passaging rescued viral replication and negative-strand synthesis, and sequencing of revertant viruses revealed reversion to the wild-type TC-83 phosphorylation capable amino acid sequences at nsP3 sites 204/5, 142, and 135. Generation of phosphomimetic mutants using aspartic acid substitutions at site 204/5 resulted in rescue of both viral replication and negative-strand RNA production, whereas phosphomimetic mutant 134/5 rescued viral replication but failed to restore negative-strand RNA levels, and phosphomimetic mutant 142 did not rescue VEEV replication. Together, these data demonstrate that IKKβ can phosphorylate VEEV nsP3 at sites 204/5, 142, and 134/5, and suggest that phosphorylation is essential for negative-strand RNA synthesis at site 204/5, but may be important for infectious particle production at site 134/5.

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Structure and sequence requirements for RNA capping at the Venezuelan Equine Encephalitis Virus RNA 5'-end

Journal of Virology ◽

10.1128/jvi.00777-21 ◽

2021 ◽

Author(s):

Oney Ortega Granda ◽

Coralie Valle ◽

Ashleigh Shannon ◽

Etienne Decroly ◽

Bruno Canard ◽

...

Keyword(s):

Present Report ◽

Regulatory Element ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Structural Protein ◽

Venezuelan Equine Encephalitis ◽

Domesticated Animals ◽

Acceptor Substrate ◽

Equine Encephalitis Virus ◽

Molecular Features

Venezuelan equine encephalitis virus (VEEV) is a re-emerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5'-end. The capping process is performed by the non-structural protein nsP1, which bears methyl and guanylyltransferases activities. The capping reaction starts by the methylation of GTP. The generated m7GTP is complexed to the enzyme to form a m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5’-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA,C, or U. We show that capping is more efficient on small RNA molecules whereas RNA longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short conserved stem loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, the re-emergence and the expansion of alphaviruses (genus of the family Togaviridae) is a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date no efficient vaccine nor safe antivirals are available for human use. VEEV non structural protein 1 (nsP1) is the viral capping enzyme characteristic of the alphavirus genus. NsP1 catalyses methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.

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Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells

Antiviral Research ◽

10.1016/j.antiviral.2016.04.008 ◽

2016 ◽

Vol 131 ◽

pp. 49-60 ◽

Cited By ~ 14

Author(s):

Moushimi Amaya ◽

Taryn Brooks-Faulconer ◽

Tyler Lark ◽

Forrest Keck ◽

Charles Bailey ◽

...

Keyword(s):

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Structural Protein ◽

Venezuelan Equine Encephalitis ◽

Rna Helicases ◽

Equine Encephalitis Virus ◽

Infected Cells

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

International Journal of Molecular Sciences ◽

10.3390/ijms21207686 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7686 ◽

Cited By ~ 1

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.

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Molecular and cellular evidence of natural Venezuelan equine encephalitis virus infection in frugivorous bats in Colombia

Veterinary World ◽

10.14202/vetworld.2020.495-501 ◽

2020 ◽

Vol 13 (3) ◽

pp. 495-501 ◽

Cited By ~ 2

Author(s):

Camilo Guzmán ◽

Alfonso Calderón ◽

Teresa Oviedo ◽

Salim Mattar ◽

José Castañeda ◽

...

Keyword(s):

Natural Infection ◽

Cross Sectional Study ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Molecular Evidence ◽

Structural Protein ◽

Venezuelan Equine Encephalitis ◽

Cross Sectional ◽

Equine Encephalitis Virus ◽

Two Samples

Background and Aim: Venezuelan equine encephalitis virus (VEEV) is an alphavirus that causes encephalitis with a high impact on public health in Latin America. However, only in Guatemala, Trinidad and Tobago, and Mexico have found antibodies in VEEV in bats, using immunohistochemistry, the sensitivity and specificity are improved; thus, it is better for demonstrating natural infection in bats as potential hosts. This study aimed to determine the presence of VEEV in tissues of frugivorous bats. Materials and Methods: A prospective descriptive cross-sectional study with a non-probabilistic sampling was carried out in 12 localities of Córdoba and Sucre area of the Colombian Caribbean. Two hundred and eighty-six bats were captured using fog nets, and the specimens according to taxonomic keys were classified. According to the Ethics Committee of the University of Córdoba, the bats were treated with analgesics and anesthetics. Blood samples were taken and then euthanized to obtain tissues and organs which were preserved in liquid N2 at –196°C. A portion of each organ was fixed in 10% buffered formalin for the detection of antigens by immunohistochemistry. Several pathological anatomy analyses were performed to determine the histological characteristics of tissue lesions of frugivorous bats naturally infected with the VEEV. Results: Of the 286 bats captured, 23 species were identified. In samples of the brain, spleen, and lung of two frugivorous bats (2/286=0.70%) Artibeus planirostris and Sturnira lilium, the presence of VEEV was confirmed by immunohistochemistry. Conclusion: A fragment of the nsP4 non-structural protein gene corresponding to the alphavirus was amplified. Two samples were positive (2/286=0.70%) in frugivorous bats; A. planirostris (code GenBank: MG820274) and S. lilium (code GenBank: MG820275). The present study showed the first molecular evidence and cellular evidence (histopathology and immunohistochemistry) of natural VEEV infection in frugivorous bats in Colombia; these bats could be a host of this zoonosis.

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Resveratrol Inhibits Venezuelan Equine Encephalitis Virus Infection by Interfering with the AKT/GSK Pathway

Plants ◽

10.3390/plants10020346 ◽

2021 ◽

Vol 10 (2) ◽

pp. 346

Author(s):

Caitlin W. Lehman ◽

Kylene Kehn-Hall ◽

Megha Aggarwal ◽

Nicole R. Bracci ◽

Han-Chi Pan ◽

...

Keyword(s):

Antiviral Activity ◽

Fluorescent Protein ◽

Glycogen Synthase ◽

Venezuelan Equine Encephalitis Virus ◽

Encephalitis Virus ◽

Host Cells ◽

Dynamics Simulation ◽

Luciferase Reporter ◽

Venezuelan Equine Encephalitis ◽

Equine Encephalitis Virus

The host proteins Protein Kinase B (AKT) and glycogen synthase kinase-3 (GSK-3) are associated with multiple neurodegenerative disorders. They are also important for the replication of Venezuelan equine encephalitis virus (VEEV), thereby making the AKT/GSK-3 pathway an attractive target for developing anti-VEEV therapeutics. Resveratrol, a natural phytochemical, has been shown to substantially inhibit the AKT pathway. Therefore, we attempted to explore whether it exerts any antiviral activity against VEEV. In this study, we utilized green fluorescent protein (GFP)- and luciferase-encoding recombinant VEEV to determine the cytotoxicity and antiviral efficacy via luciferase reporter assays, flow cytometry, and immunofluorescent assays. Our results indicate that resveratrol treatment is capable of inhibiting VEEV replication, resulting in increased viability of Vero and U87MG cells as well as reduced virion production and viral RNA contents within host cells for at least 48 h with a single treatment. Furthermore, the suppression of apoptotic signaling adaptors, caspase-3, caspase-7, and annexin V may also be implicated in resveratrol-mediated antiviral activity. We found that decreased phosphorylation of the AKT/GSK-3 pathway, mediated by resveratrol, can be triggered during the early stages of VEEV infection, suggesting that resveratrol disrupts the viral replication cycle and consequently promotes cell survival. Finally, molecular docking and dynamics simulation studies revealed that resveratrol can directly bind to VEEV glycoproteins, which may interfere with virus attachment and entry. In conclusion, our results suggest that resveratrol exerts inhibitory activity against VEEV infection and upon further modification could be a useful compound to study in neuroprotective research and veterinary sciences.

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