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

Viruses ◽  
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.

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.

scholarly journals Mutations in Hypervariable Domain of Venezuelan Equine Encephalitis Virus nsP3 Protein Differentially Affect Viral Replication

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Chetan D. Meshram ◽  
Aaron T. Phillips ◽  
Tetyana Lukash ◽  
Nikita Shiliaev ◽  
Elena I. Frolova ◽  
...  
Keyword(s):  
Viral Replication ◽  
Family Members ◽  
Sh3 Domain ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Cellular Proteins ◽  
Venezuelan Equine Encephalitis ◽  
Host Proteins ◽  
Equine Encephalitis Virus

ABSTRACT Venezuelan equine encephalitis virus (VEEV) is one of the important human and animal pathogens. It forms replication enzyme complexes (RCs) containing viral nonstructural proteins (nsPs) that mediate the synthesis of virus-specific RNAs. The assembly and associated functions of RC also depend on the presence of a specific set of host proteins. Our study demonstrates that the hypervariable domain (HVD) of VEEV nsP3 interacts with the members of the FXR family of cellular proteins and also binds the Src homology 3 (SH3) domain-containing proteins CD2AP and SH3KBP1. Interactions with FXR family members are mediated by the C-terminal repeating peptide of HVD. A single short, minimal motif identified in this study is sufficient for driving efficient VEEV replication in the absence of HVD interactions with other host proteins. The SH3 domain-containing proteins bind to another fragment of VEEV HVD. They can promote viral replication in the absence of FXR-HVD interactions albeit less efficiently. VEEV replication can be also switched from an FXR-dependent to a chikungunya virus-specific, G3BP-dependent mode. The described modifications of VEEV HVD have a strong impact on viral replication in vitro and pathogenesis. Their effects on viral pathogenesis depend on mouse age and the genetic background of the virus. IMPORTANCE The replication of alphaviruses is determined by specific sets of cellular proteins, which mediate the assembly of viral replication complexes. Some of these critical host factors interact with the hypervariable domain (HVD) of alphavirus nsP3. In this study, we have explored binding sites of host proteins, which are specific partners of nsP3 HVD of Venezuelan equine encephalitis virus. We also define the roles of these interactions in viral replication both in vitro and in vivo. A mechanistic understanding of the binding of CD2AP, SH3KBP1, and FXR protein family members to VEEV HVD uncovers important aspects of alphavirus evolution and determines new targets for the development of alphavirus-specific drugs and directions for viral attenuation and vaccine development.

scholarly journals Identification of Quinolinones as Antivirals Against Venezuelan Equine Encephalitis Virus

2021 ◽  
Author(s):  
Nicole N. Haese ◽  
Nicholas A. May ◽  
Sharon Taft-Benz ◽  
Omar Moukha-Chafiq ◽  
Nikhil Madadi ◽  
...  
Keyword(s):  
Viral Replication ◽  
Inhibitory Activity ◽  
Reverse Genetics ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Resistance Mutations ◽  
Nsp2 Gene ◽  
Equine Encephalitis Virus ◽  
Stalk Domain

Venezuelan equine encephalitis virus (VEEV) is a re-emerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time of addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that endowed VEEV resistance to this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of CHIKV to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses, and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

scholarly journals Protein Phosphatase 1α Interacts with Venezuelan Equine Encephalitis Virus Capsid Protein and Regulates Viral Replication through Modulation of Capsid Phosphorylation

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Brian D. Carey ◽  
Tatiana Ammosova ◽  
Chelsea Pinkham ◽  
Xionghao Lin ◽  
Weidong Zhou ◽  
...  
Keyword(s):  
Viral Replication ◽  
Protein Phosphatase ◽  
Rift Valley Fever Virus ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Valley Fever ◽  
Regulatory Subunits ◽  
Equine Encephalitis Virus ◽  
Hiv 1

ABSTRACTProtein phosphatase 1 (PP1) is a serine/threonine phosphatase which has been implicated in the regulation of a number of viruses, including HIV-1, Ebolavirus, and Rift Valley fever virus. Catalytic subunits of PP1 (PP1α, PP1β, and PP1γ) interact with a host of regulatory subunits and target a wide variety of cellular substrates through a combination of short binding motifs, including an RVxF motif present in the majority of PP1 regulatory subunits. Targeting the RVxF-interacting site on PP1 with the small molecule 1E7-03 inhibits HIV-1, Ebolavirus, and Rift Valley fever virus replication. In this study, we determined the effect of PP1 on Venezuelan equine encephalitis virus (VEEV) replication. Treatment of VEEV-infected cells with 1E7-03 decreased viral replication by more than 2 logs (50% effective concentration [EC50] = 0.6 μM). 1E7-03 treatment reduced viral titers starting at 8 h postinfection. Viral replication was also decreased after treatment with PP1α-targeting small interfering RNA (siRNA). Confocal microscopy demonstrated that PP1α shuttles toward the cytosol during infection with VEEV and that PP1α colocalizes with VEEV capsid. Coimmunoprecipitation experiments confirmed VEEV capsid interaction with PP1α. Furthermore, immunoprecipitation and mass spectrometry data showed that VEEV capsid is phosphorylated and that phosphorylation is moderated by PP1α. Finally, less viral RNA is associated with capsid after treatment with 1E7-03. Coupled with data showing that 1E7-03 inhibits several alphaviruses, this study indicates that inhibition of the PP1α RVxF binding pocket is a promising therapeutic target and provides novel evidence that PP1α modulation of VEEV capsid phosphorylation influences viral replication.IMPORTANCEVenezuelan equine encephalitis virus (VEEV) causes moderate flu-like symptoms and can lead to severe encephalitic disease and potentially death. There are currently no FDA-approved therapeutics or vaccines for human use, and understanding the molecular underpinning of host-virus interactions can aid in the rational design of intervention strategies. The significance of our research is in identifying the interaction between protein phosphatase 1 (PP1) and the viral capsid protein. This interaction is important for viral replication, as inhibition of PP1 results in decrease viral replication. Inhibition of PP1 also inhibited multiple biomedically important alphaviruses, indicating that PP1 may be a potential therapeutic target for alphavirus-induced disease.

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

Plants ◽  
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.

scholarly journals Spatial Dispersion of Adult Mosquitoes (Diptera: Culicidae) in a Sylvatic Focus of Venezuelan Equine Encephalitis Virus

2001 ◽  
Vol 38 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Wilmer Méndez ◽  
Jonathan Liria ◽  
Juan-Carlos Navarro ◽  
Carmen Z. García ◽  
Jerome E. Freier ◽  
...  
Keyword(s):  
Spatial Dispersion ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus

Congenital cerebral and ocular malformations induced in rhesus monkeys by Venezuelan Equine Encephalitis virus

Teratology ◽  
1977 ◽  
Vol 16 (3) ◽  
pp. 285-295 ◽  
Author(s):  
W. T. London ◽  
Neil H. Levitt ◽  
Stephen G. Kent ◽  
Vernon G. Wong ◽  
John L. Sever
Keyword(s):  
Rhesus Monkeys ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus

scholarly journals The Role of IKKβ in Venezuelan Equine Encephalitis Virus Infection

PLoS ONE ◽  
2014 ◽  
Vol 9 (2) ◽  
pp. e86745 ◽  
Author(s):  
Moushimi Amaya ◽  
Kelsey Voss ◽  
Gavin Sampey ◽  
Svetlana Senina ◽  
Cynthia de la Fuente ◽  
...  
Keyword(s):  
Virus Infection ◽  
Venezuelan Equine Encephalitis Virus ◽  
Encephalitis Virus ◽  
Venezuelan Equine Encephalitis ◽  
Equine Encephalitis Virus
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