Aichi virus, bovine kobuvirus, and klassevirus 3A proteins are myristoylated.
To identify proteins that interact with the main membrane-reorganizing protein of picornaviruses, protein 3A, we undertook an unbiased screen using single-step affinity purification of C-terminally Strep-Tagged 3A proteins in 293T cells followed by mass-spectrometric peptide sequencing analysis of in-solution trypsin digests. Careful inspection of the mass spectrometry data identified a myristoylation on the N-terminal glycine of Aichi virus 3A, bovine kobuvirus 3A, and klassevirus 3A (). The activity of N
-myristoyltransferase (NMT) enzymes is strongly dependent on the first five residues, with an N-terminal glycine being absolutely required (27
). The N-terminal sequence of Aichi virus 3A is GNRVIDAE. Although algorithms such as NMT—The Myr Predictor (http://mendel.imp.ac.at/myristate/SUPLpredictor.htm
) and the ExPASy Myristoylator do not predict the N terminus of any of the kobuviruses to be NMT substrates, in vitro
experiments using human NMT1 have shown synthetic octapeptides such as GNRAAARR to be valid substrates with kinetics comparable to those of other known substrates (3
To explore the functional effects of myristoylation, N-terminal mutations of the Aichi virus 3A protein were analyzed. In Aichi virus 3A, the G1A mutation abolishes myristoylation, as do N2A and R3A, while V4A, I5A, D6A, and E8A mutants retain N-terminal myristoylation, as measured by mass spectrometry analysis of the affinity-purified protein. Mass spectra for the I5A and R3A mutants of Aichi virus 3A are shown in D and E, and unique peptide identifications for this analysis are provided in Table S3 in the supplemental material. Targeted analysis of MS spectra for affinity purifications of 3A proteins derived from coxsackieviruses B2, B3, and B5, enterovirus 71, poliovirus 1, and human rhinovirus 14 did not reveal evidence of N-terminal myristoylation, despite the fact that significant peptide counts for unmodified or acetylated N termini were recovered. The cardioviruses, theilovirus strains TMDA, BeAn, and TMGDVII, and Saffold viruses UC6 and Saf2 have serines as their start residues and thus cannot be substrates for NMTs. For all 3A affinity purification experiments, additional searches were performed for other posttranslational modifications, including phosphorylations, GlyGly signatures for ubiquitination, and broad-mass-range modifications of up to 500 atomic mass units. With the exception of N-terminal acetylation, cysteine carbamidomethylation, pyroglutamylation of glutamine, and oxidation, no other modifications were detected in these experiments.
The 3A protein was also frequently observed to run as a doublet at 15 and 17 kDa by SDS-PAGE (for an example, see A) with detection by silver staining or by anti-streptavidin tag antibody in Western blot format. In the case of Aichi virus 3A, mass-spectrometric analysis confirmed that both bands contained full-length 3A protein, the lower band comigrating with streptavidin. Despite extensive searches, no posttranslational modifications, including myristoylation, could be found that explain a mass shift in these bands, suggesting that these may be conformationally resolved forms of the protein. N-terminal and C-terminal peptides for wild-type and mutant Aichi virus 3A proteins are provided in Table S4 in the supplemental material.
Fig 2 Picornaviral 3A proteins interact differentially with PI4KIIIβ and ACBD3. (A) Strep-Tagged 3A proteins across the picornavirus family were affinity captured under fully equilibrated binding conditions and then blotted with anti-ACBD3, anti-PI4KIIIβ, (more ...) Method for determining specific interactions from mass spectrometry data.
To identify proteins that specifically interact with picornavirus 3A within the set of proteins identified by mass spectrometry, we first attempted to use a Strep-Tagged GFP as a negative control for nonspecific interactions. The combined protein identifications, across four replicate experiments for Aichi virus 3A and GFP, resulted in approximately 70 and 40 putative interacting proteins, respectively (see Tables S5 and S6 in the supplemental material). Based on these results, we determined that Strep-Tagged GFP did not adequately sample the spectrum of nonspecific interactions in this experimental system, consistent with observations by other groups (6
Therefore, we chose instead to perform a comprehensive analysis of background proteins, assessing their ability to interact with multiple unrelated viral proteins, consisting of 91 unique non-3A picornaviral bait proteins assayed in 293 individual experiments (see Materials and Methods). The set of background interacting proteins was then used to derive the specificity for virus bait-host protein interactions, ranked using Z-scores. To minimize false positives, we report interactions that pass a highly conservative Z-score threshold of 10 and whose preys are represented by a minimum of two peptides in at least two biological replicates. To further strengthen confidence in the analysis, Aichi virus 3A affinity purifications were compared against those of 3A proteins from 15 diverse picornaviruses. The most specific protein interaction partners for each 3A protein were ranked using the Z-score metric and resulted in a refined list of candidate interactions (). This method of scoring interactions was in part confirmed by the top ranking of GBF1 for poliovirus and coxsackievirus 3A proteins. Prior to this study, GBF1 was the only confirmed protein to copurify with any picornaviral 3A. The complete table of Z-scores for all identified interacting proteins across all picornaviral 3A proteins tested is reported in Table S7 in the supplemental material.
Highest specificity picornavirus 3A-human protein-protein interactions, ranked by Z-scorea
Affinity purification-MS (AP-MS) of C-terminally Strep-Tagged picornaviral 3A proteins copurifies PI4KIIIβ and ACBD3.
The top-ranked protein identified in affinity purifications with both Aichi virus 3A and bovine kobuvirus 3A was PI4KIIIβ (). PI4KIIIβ interaction with Aichi virus 3A and bovine kobuvirus 3A was confirmed by Western blot (A). Intriguingly, one peptide to PI4KIIIβ was found in one replicate of coxsackievirus B5 affinity purification by MS, and a weak positive result by Western blot (A) was also observed. To investigate whether other 3A proteins such as poliovirus 3A might still interact with PI4KIIIβ more transiently, affinity purifications for selected enteroviral 3A proteins were repeated under more rapid kinetic conditions, using short binding and washing steps and capture on magnetic StrepTactin beads (6
). Using this more rapid procedure, PI4KIIIβ was detected by affinity purification with 3A proteins from poliovirus, human rhinovirus 14, and coxsackievirus B3 (B). We note that the 3A protein from rhinovirus 14 also captured GBF1, consistent with HRV sensitivity to brefeldin A (15
The second-ranking protein identified in the Aichi virus 3A affinity purifications was acyl-CoA binding domain protein 3 (ACBD3), also known as Golgi complex-associated protein GCP60 (). This protein was also affinity purified specifically by the 3A proteins of multiple picornaviruses, including poliovirus, Aichi virus, bovine kobuvirus, porcine kobuvirus, human rhinovirus 14, and coxsackie B viruses. Although the 3A protein of EV71 did not copurify with ACBD3 under these conditions, we note that it did copurify with a different acyl-CoA binding protein, ACAD9. Although it was not detectable by Western blotting (A), a single peptide for ACBD3 was detected in an EV71 3A protein AP-MS experiment using rapid capture and wash steps on StrepTactin Sepharose beads (data not shown); thus, we cannot exclude interaction of EV71 with ACBD3. ACBD3 is a Golgi resident protein that has been implicated in multiple cell signaling systems, including Golgi complex maintenance, steroidogenesis, and apoptosis (11
). Interaction between ACBD3 and picornaviral 3A proteins was confirmed by Western blotting (A). Furthermore, Aichi virus 3A and PI4KIIIβ were both immunoprecipitated by anti-ACBD3 antibody in Aichi virus 3A-transfected 293T cells, as detected by mass spectrometry (see Table S8 in the supplemental material). However, in the absence of Aichi virus 3A, endogenous PI4KIIIβ and ACBD3 did not coprecipitate with each other, suggesting that 3A specifically stabilizes the complex containing both of these proteins (see Table S8).
To test whether ACBD3 and PI4KIIIβ have direct interactions in the absence of Aichi virus 3A, C-terminally Strep-Tagged ACBD3 was transiently transfected and affinity purified using the rapid binding and washing protocol with and without a series of 3A proteins from entero- and kobuviruses. In the absence of any transfected 3A proteins, PI4KIIIβ was found to copurify with ACBD3 (, empty vector lane), indicating that the interaction between ACBD3 and PI4KIIIβ does not require 3A. Affinity purification of ACBD3 in the presence of 3A proteins captured 3A from poliovirus, coxsackievirus B3, human rhinovirus 14, Aichi virus, and bovine kobuvirus. The only exception was EV71, consistent with the reciprocal affinity purification experiments discussed above. Surprisingly, a band consistent with a complex containing Strep-Tag-ACBD3 and Aichi virus-3A-Flag was observed (, Aichi virus 3A lane) despite standard denaturing gel running conditions. Taken together, the results of this reciprocal affinity capture experiment imply direct interaction between ACBD3, PI4KIIIβ, and multiple picornaviruses.
Fig 3 Affinity purification of Strep-Tagged ACBD3. Strep-Tagged ACBD3 and FLAG-tagged enterovirus and kobuvirus 3A proteins were transiently cotransfected into 293T cells, and ACBD3 complexes were affinity captured under rapid kinetic conditions. Samples were (more ...)
Alignment of all Aichi virus 3A sequences in GenBank indicated >90% amino acid identity among the five sequences available, including 100% conservation in the N-terminal half of the protein.
Site-directed mutagenesis of Aichi virus 3A identifies residues required for interaction with PI4KIIIβ and ACBD3.
To identify the critical residues for the interaction between Aichi virus 3A, PI4KIIIβ, and ACBD3, we employed alanine scanning of the 95-amino-acid Aichi virus 3A protein. In total, all 87 nonalanine residues were converted to alanine with a focus on single-site mutants on the N-terminal half, where all Aichi virus 3A sequences in GenBank are 100% conserved, in addition to multisite mutants on the C terminus (A). Of 87 positions mutated, approximately 20 residues (B), clustered at the N terminus, severely reduced or abolished copurification of PI4KIIIβ (<10% of wild type, normalized to expression of the 3A protein in each experiment). In particular, the mutations R3A, I5A, NR2AA, NRV2AAA, and NRVI2AAAA abolished PI4KIIIβ and ACBD3 interaction or reduced the amount by more than 90%. Although the R3A and G1A mutations both eliminate the N-terminal myristoylation of Aichi virus 3A, the lack of myristoylation does not account for the loss of PI4KIIIβ and ACBD3 interaction, since N2A, which also eliminates the N-terminal myristoylation, has no effect on copurification of either of these proteins (A). While 21 mutations could eliminate PI4KIIIβ association without affecting association of ACBD3 with Aichi virus 3A, all mutations that had a negative impact on ACBD3 association also severely reduced or eliminated PI4KIIIβ association. These results support the hypothesis that PI4KIIIβ association with Aichi virus 3A requires ACBD3 and also imply that 3A association with ACBD3 either enhances or stabilizes the interaction with PI4KIIIβ.
Fig 4 Site-directed mutagenesis of Aichi virus 3A identifies residues required for interaction with PI4KIIIβ and ACBD3. (A) Alanine scanning of individual and multiple residues in Aichi virus 3A was performed. Mutated Aichi virus 3A was transiently (more ...) Chemical and genetic inhibition of PI4KIIIβ blocks Aichi virus replication.
To assess whether the interaction between Aichi virus 3A and PI4KIIIβ was of functional importance for viral replication, we measured a Renilla
luciferase replicon Aichi virus construct in the presence of chemical and genetic inhibition of PI4KIIIB. PIK93 is a small-molecule inhibitor of PI4Kα and PI4KIIIβ (19
). It has previously been shown to block the replication of poliovirus and hepatitis C virus replication with 50% effective concentrations (EC50
) of 0.14 and 1.9 μM, respectively (1
). The addition of 0.5 μM and 1.0 μM PIK93 resulted in a dose-dependent inhibition of Aichi virus replication similar to the dose-dependent decrease observed with poliovirus (A and B).
Fig 5 Chemical and genetic inhibition of PI4KIIIβ decreases Aichi virus replication. PIK93, an inhibitor of PI4KIIIβ, demonstrates a dose-dependent block in poliovirus (A) and Aichi virus replication (B). Genetic inhibition of PI4KIIIβ (more ...)
Stable shRNAs were used to reduce or nearly eliminate PI4KIIIβ expression in 293T cells, using previously published shRNAs (C and D) (4
). PI4KIIIβ mRNA transcript abundance was reduced by up to 98% relative to the expression of the ribosome gene RPL19 (E). The shRNA-dependent knockdown PI4KIIIβ protein expression was also confirmed by Western blotting (F). Only the most potent shRNA construct inhibited Aichi virus and poliovirus replication, while incomplete knockdown of PI4KIIIβ did not significantly impact Aichi virus or poliovirus replication (C and D).
While siRNA knockdown of PI4KIIIβ completely abolished Aichi virus and poliovirus replication, knockdown of ACBD3 demonstrated significantly reduced replication in both viruses (A and B). Although Aichi virus replication has been reported to be insensitive to brefeldin A (35
), an inhibitor of GBF1/Arf1, we were surprised to find that siRNA knockdown of GBF1 also abolishes Aichi virus replication (B). Interestingly, the siRNA knockdown of GBF1 resulted in a loss of PI4KIIIβ, similar to what was achieved with a directed siRNA knockdown of PI4KIIIβ (C). While these results demonstrate a requirement for GBF1, it is possible that the replication defect is actually due to a loss of PI4KIIIβ indirectly caused by a loss of GBF1. These results support the hypothesis that the presence and activity of PI4KIIIβ are essential for Aichi virus replication, similar to what has been shown previously for poliovirus (17
). These data also support the hypothesis that ACBD3 is functionally important for picornavirus replication, presumably by facilitating the interaction with PI4KIIIβ.
Fig 6 Genetic inhibition of physically interacting genes reduces viral replication. siRNA knockdown of GBF1, PI4KIIIβ, and ACBD3 reduces poliovirus (A) and Aichi virus (B) replicon growth in HeLa cells relative to control siRNA. RLU, relative light (more ...) Reduced recruitment of PI4KIIIβ correlates with delayed or altered replication kinetics of Aichi virus replicons.
To further assess the requirement of the association between Aichi virus 3A and PI4KIIIβ for viral replication, we tested viral replication after replacing the wild-type 3A sequence with a series of point mutations based on our affinity purification results. No replication above background was measureable in the context of the Aichi virus replicon with the E11A mutation, which significantly disrupted copurification with PI4KIIIβ and reduced, but did not eliminate, ACBD3 association (A). Aichi virus with the G1A mutation also demonstrated no replication, though this is likely due to disruption of the P2-P3 3C proteolytic cleavage site. Myristoylation had a minimal impact on replication, as the N2A mutant had near-wild-type levels of replication, while replication of the R3A mutant was slightly delayed (B). Delayed replication was observed with the NR2AA, NRV2AAA, NRVI2AAAA, I5A, I12A, L20A, L21A, M24A, and HH26AA mutations, which significantly reduced copurification of PI4KIIIβ by >90%, though several of the mutations retained the same maximal level of replication as the wild-type Aichi virus replicon (C to F). The E22A mutation that retained wild-type PI4KIIIβ binding demonstrated replication kinetics that were slightly delayed but comparable to those of the wild-type virus (G). The P59A mutation, which appeared to stimulate PI4KIIIβ copurification (A), resulted in a significant delay in replication, yet the virus continued to produce luciferase signal for more than 10 h posttransfection (G).
Fig 7 Reduced recruitment of PIKIIIβ correlates with delayed replication kinetics of Aichi virus replicons. 3A mutants were cloned into Aichi virus replicon and examined for replication efficiency. (A) The Aichi virus 3A E11A mutant is unable to replicate. (more ...)
To ascertain whether the delayed replication in viruses with mutations in 3A that inhibited PI4KIIIβ association was due to the reduced PI4KIIIβ association, we examined the EC50 of PIK93 in these mutant viruses compared to wild-type virus. PIK93 had an EC50 of 0.60 μM at 330 min posttransfection in the wild-type Aichi virus replicon. Interestingly, lower concentrations of PIK93 did not reduce the total amount of viral replication but only delayed viral replication in a dose-dependent fashion (A). However, the EC50 of PIK93 was reduced more than 2-fold to 0.24 μM in the I5A mutant and almost 20-fold to 0.03 μM in the NRVI2AAAA mutant (B). These results support the notion that physical association of PI4KIIIβ with Aichi virus 3A via ACBD3 is required for replication.
Fig 8 Aichi virus replicons with 3A mutants with reduced ability to recruit PI4KIIIβ are sensitized to PIK93 inhibition. (A) The EC50 for PIK93 against wild-type Aichi virus replicon was 0.60 μM at 330 min posttransfection. PIK93 inhibition (more ...)