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.
Highlights
Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, is a member of the Togaviridae family of RNA viruses
We identify IKKβ as the first reported host kinase capable of phosphorylating VEEV non-structural protein 3 (nsP3) on residues 204/5, 142, and 134/5 and demonstrate that phosphorylation of residues 204/5 is essential for negative-strand synthesis while phosphorylation of residues 134/5 may be important for infectious particle production
We identified potential sites phosphorylated on VEEV nsP3 in silico (Figure 1A) using the Reference Sequence L01443 for VEEV TC-83 and the prediction software, NetPhos3.1
Summary
Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, is a member of the Togaviridae family of RNA viruses. Infection with VEEV can progress to encephalitic disease that is fatal in ~1% of cases [1,2,3] whereas Old World alphavirus infection The VEEV genome consists of a positive-sense, single stranded RNA molecule ~11.4 kb in length that encodes two polyproteins [1,5]. The non-structural polyprotein consists of the four non-structural proteins nsP1-4, that drive viral RNA replication in concert with a range of host proteins [9]. The structural polyprotein is cleaved into the viral capsid and glycoproteins, which are responsible for packaging genomes into progeny virions [1,10]. While multiple activities of nsP1, 2, and 4 have been described, the activities of nsP3 have only recently begun to be elucidated [9]
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