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

Abstract

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.

Keywords: IKK complex; IKKβ kinase assay; Venezuelan equine encephalitis virus; mutations; negative-strand; non-structural protein 3; phosphomimetics; phosphorylation; replication-deficient; revertants; sequencing.

Figure 1

VEEV nsP3 is phosphorylated and inhibition of IKKβ reduces nsP3 phosphorylation and viral replication levels. (A) VEEV nsP3 amino acid residues predicted to be phosphorylated by host kinases were identified using the NetPhos 3.1 server and VEEV TC-83 L01443 sequence obtained from NCBI. (B) pcDNA 3.1(+) empty vector or pCAGGS plasmid expressing N-terminus HA-tagged wild-type VEEV ZPC738-nsP3 were transfected in U-87MG cells in the presence or absence of the IKKβ inhibitor BAY-11-7082 (1 µM) for 24 h. Lysates were probed for phospho-serine, phospho-threonine, phospho-tyrosine, or HA-tag and signals were calculated and normalized as described in Section 2. Western blot and graphical data are representative of three independent experiments (n = 3). ND represents undetectable virus (C) U-87MG cells were treated with DMSO or the IKKβ inhibitor BAY-11-7082 (1 µM) for 2 h, subsequently infected with TC-83 at MOI of 0.1 in triplicate for 1 h and conditioned media containing BAY-11-7082 or standard media was replaced after removal of virus. At 2, 6, 16 hpi, RNA was collected, and RT-PCR performed as described in Section 2. Graph represents data from two independent experiments performed in triplicates (n = 6). ** p < 0.0021 and **** p < 0.0001, ns, not significant.

Figure 2

IKKβ activity directly phosphorylates VEEV nsP3. (A) Schematic of Kinase HotSpot Assay performed by Reaction Biology Corp. as described in Section 2. Illustration was generated using Biorender. (B) VEEV TC-83 nsP3 was expressed and purified from a bacterial expression system as described in Section 2. A cell-free, in vitro assay of IKKβ enzyme vs. purified VEEV nsP3 measured the amount of ³³P-γ-ATP transferred onto substrate. GAPDH was included as a negative control substrate and ‘IKKtide’, a small validated peptide, was included as a positive control. Graph is representative of the average ³³P counts measured for duplicate reactions of substrate, corrected for purity, and incubated with 200 nM IKKβ for 1 h (n = 2). (C) WT or IKKβ^−/− MEF cells were infected with TC-83 at MOI of 2 for 1 h and media was replaced after removal of virus. At 6 hpi, total protein lysates were obtained and subjected to LC-MS/MS. Mass spectra were fitted against NCBI reference sequence L01443 for VEEV TC-83 nsP3. (D) U-87MG cells were untreated or pre-treated with 1 µM BAY-11-7082 for 2 h, infected with TC-83 at MOI of 1 for 1 h and conditioned media containing 1 µM BAY-11-7082 or standard media was replaced after removal of virus. At 8 hpi, total protein lysates were obtained and subjected to LC-MS/MS. Mass spectra were fitted against NCBI reference sequence NP_740698.1 for putative VEEV nsP3. Phosphorylated amino acid residue sites detected on VEEV nsP3 by LC-MS/MS are listed in panels (B,C). * p < 0.0332 and **** p < 0.0001.

Figure 3

VEEV nsP3 mutants 204/5, 142, and 134/5 are replication-deficient in both TC-83 and TrD strains. (A) Graphic illustration of VEEV nsP3 domains. The locations of alanine substitutions for each generated VEEV mutant are represented by their amino acid residue numbers. (B) WT and mutant viruses were grown as described in Section 2 and viral titers were measured via plaque assay. ND represents undetectable virus. Mutants 204/5 and 142 were titered in triplicate from four independent experiments (n = 12). All other mutants were titered in triplicate from two independent experiments (n = 6). (C) WT and mutant viruses were grown as described in Section 2 and titers were measured via plaque assay. Graph is representative of one independent experiment (n = 3). (D) C6/36 mosquito cells were infected in triplicate at MOI of 1 (wild-type and mutant 134/5) or undiluted virus (mutants 204/5 and 142). At 24 hpi, supernatants were collected and plaque assay was performed as described in Section 2. Graph is representative of two independent experiments (n = 6). (E) Vero cells were infected in triplicate with VEEV TC-83 nsP3 mutants using undiluted virus or diluted 1:1. At 24 hpi, supernatants were collected, and luciferase assay was performed as described in Section 2. Graph is representative of two independent experiments (n = 6). * p < 0.0332, ** p < 0.0021, *** p < 0.0002, and **** p < 0.0001, ns, not significant.

Figure 4

Negative-strand synthesis is curtailed during replication-deficient mutant infection and IKKβ phosphorylates these VEEV nsP3 sites. Vero cells were infected in triplicate at MOI of 1 (wild-type and mutant 134/5) or undiluted virus (mutants 204/5 and 142). At 2, 6 and 16 hpi, RT-PCR to measure positive and negative strand levels was performed as described in Section 2. (A) Expression levels of capsid. (B) Expression levels of nsP3. (C) Expression levels of negative-strand RNA. Graphs are representative of two independent experiments (n = 6). (D) VEEV TC-83 nsP3 wild-type and mutant proteins were expressed and purified from a bacterial expression system as described in Section 2. A cell-free, in vitro assay of IKK. β enzyme vs. purified VEEV nsP3 measured the amount of ³³P-γ-ATP transferred onto substrate. GAPDH was included as an inert negative control substrate and ‘IKKtide’, a small validated peptide, was included as a positive control. Graph is representative of the average ³³P counts measured for duplicate reactions of substrate, corrected for purity, and incubated with 200 nM IKKβ for 1 h (n = 2). * p < 0.0332, *** p < 0.0002, and **** p < 0.0001.

Figure 5

Serial passaging of replication-deficient VEEV nsP3 mutants generates revertant viruses competent for negative-strand synthesis. (A) Schematic of serial passaging performed using replication-deficient mutants as described in Section 2. (B) Viral supernatants from serial passages 11–13 for mutants 204/5 and 142 and passage 5 for mutant 134/5 were used to infect Vero cells to evaluate cytopathic effect (CPE) as compared to wild-type TC-83 and mock infected cells. (C) Viral supernatants from serial passaging were titered in triplicate. (D) Negative-strand synthesis levels were measured by RT-PCR as described in Section 2. Graphs are representative of duplicate serial passages prepared in triplicate (n = 6). **** p < 0.0001.

Figure 6

Serial passaging of replication-deficient viruses restores wild-type TC-83 amino acids at positions 204/5, 142, and 135. cDNA specific to nsP3 and 3′UTR was generated, amplified, and sequenced as described in Section 2. (A) Protein sequence alignment of nsP3 as compared to wild-type TC-83. ° Indicates the wild-type TC-83 sequence with inclusion of all alanine residues originally substituted in each mutant at sites of interest. * and ** indicate sites of reversion and mutational changes in nsP3. Red boxes highlight original alanine substitution sites and black boxes indicate amino acid changes at additional sites in nsP3. (B) Amino acid and nucleotide genomic changes and chromatogram traces observed for each revertant mutant in nsP3. (C) Nucleotide sequence alignment and chromatogram traces of CSE for revertant viruses as compared to wild-type TC-83. (D) Protein sequence alignment of nsP3 for representative OW and NW alphaviruses. Green boxes highlight conserved amino acid residues of interest.

Figure 7

Phosphomimetic mutations at nsP3 sites 204/5 and 134/5 rescue VEEV replication. Vero cells were untreated or pretreated with 10 µM BAY-11-7082 for 2 h, subsequently infected with VEEV TC-83 nsP3 alanine or aspartic acid mutants in triplicate for 1 h, and conditioned media containing BAY-11-7082 or standard media was replaced after removal of virus. Cells were infected at MOI of 1 (wild-type and mutants 204D/5D, 134A/5A, 134D/5D) or undiluted virus (mutants 204A/5A, 142A, 142D). At 24 hpi, supernatants were collected and cellular lysates obtained. (A) Plaque assay using viral supernatants and (B) intracellular luciferase assay were performed as described in Section 2. ND represents undetectable virus. (C) Vero cells were infected with VEEV TC-83 nsP3 alanine or aspartic acid mutants in triplicate at MOI of 1 (wild-type and mutants 204D/5D, 134A/5A, 134D/5D) or undiluted virus (mutants 204A/5A, 142A, 142D). At 6 hpi, RT-PCR to measure negative strand levels was performed in triplicate as described in Section 2. Graphs are representative of two independent experiments (n = 6). * p < 0.0332, ** p < 0.0021, *** p < 0.0002, and **** p < 0.0001, ns, not significant.