Phosphorylated VP30 of Marburg Virus Is a Repressor of Transcription

Abstract

The filoviruses Marburg virus (MARV) and Ebola virus (EBOV) cause hemorrhagic fever in humans and nonhuman primates, with high case fatality rates. MARV VP30 is known to be phosphorylated and to interact with nucleoprotein (NP), but its role in regulation of viral transcription is disputed. Here, we analyzed phosphorylation of VP30 by mass spectrometry, which resulted in identification of multiple phosphorylated amino acids. Modeling the full-length three-dimensional structure of VP30 and mapping the identified phosphorylation sites showed that all sites lie in disordered regions, mostly in the N-terminal domain of the protein. Minigenome analysis of the identified phosphorylation sites demonstrated that phosphorylation of a cluster of amino acids at positions 46 through 53 inhibits transcription. To test the effect of VP30 phosphorylation on its interaction with other MARV proteins, coimmunoprecipitation analyses were performed. They demonstrated the involvement of VP30 phosphorylation in interaction with two other proteins of the MARV ribonucleoprotein complex, NP and VP35. To identify the role of protein phosphatase 1 (PP1) in the identified effects, a small molecule, 1E7-03, targeting a noncatalytic site of the enzyme that previously was shown to increase EBOV VP30 phosphorylation was used. Treatment of cells with 1E7-03 increased phosphorylation of VP30 at a cluster of phosphorylated amino acids from Ser-46 to Thr-53, reduced transcription of MARV minigenome, enhanced binding to NP and VP35, and dramatically reduced replication of infectious MARV particles. Thus, MARV VP30 phosphorylation can be targeted for development of future antivirals such as PP1-targeting compounds. IMPORTANCE The largest outbreak of MARV occurred in Angola in 2004 to 2005 and had a 90% case fatality rate. There are no approved treatments available for MARV. Development of antivirals as therapeutics requires a fundamental understanding of the viral life cycle. Because of the close similarity of MARV to another member of Filoviridae family, EBOV, it was assumed that the two viruses have similar mechanisms of regulation of transcription and replication. Here, characterization of the role of VP30 and its phosphorylation sites in transcription of the MARV genome demonstrated differences from those of EBOV. The identified phosphorylation sites appeared to inhibit transcription and appeared to be involved in interaction with both NP and VP35 ribonucleoproteins. A small molecule targeting PP1 inhibited transcription of the MARV genome, effectively suppressing replication of the viral particles. These data demonstrate the possibility developing antivirals based on compounds targeting PP1.

FIG 1

Analysis of MARV VP30 phosphorylation by LC-MS/MS. (A) Purification of Flag-tagged MARV VP30 for LC-MS/MS analysis. Cells were transfected with a VP30-expressing plasmid, untreated (lane 1) or treated with 10 μM 1E7-03 overnight (lane 2) or 0.1 μM okadaic acid (OA) for 2 h (lane 3). Lane 4, molecular weight (MW) markers. The arrow indicates the position of VP30. (B) MS/MS analysis of MARV VP30 conducted with Proteome Discoverer, version 1.4, and the SEQUEST search engine. Peptides were in-gel digested with trypsin, eluted, and subjected to MS analysis on a Thermo linear trap quadrupole (LTQ) Orbitrap XL mass spectrometer. Shown is the sequence of VP30. Peptides identified with high and low probability are shown in green and blue, respectively, and the phosphorylated residues are indicated with asterisks. (C) Identified VP30 phosphopeptides. (D to F) MS/MS spectra of selected phosphorylated and nonphosphorylated VP30 peptides: SsAESSPTNHIPR, ARPPsTFNLSKPPPPPK, and AALSLTcAGIR, as indicated. Lowercase letters indicate phosphorylation (s) or alkylation (c). Color peaks indicate matched MS/MS fragments, where green indicates precursors, and blue and red indicate y and b ions, respectively.

FIG 2

Analysis of MARV virion-associated VP30 phosphorylation by LC-MS/MS. (A) Purification of MARV for LC-MS/MS analysis. (B) MS/MS analysis of MARV VP30 conducted with Proteome Discoverer, version 1.4, and the SEQUEST search engine. Peptides were in-gel digested with trypsin, eluted, and subjected to MS analysis on a Thermo LTQ Orbitrap XL mass spectrometer. Shown is the sequence of VP30. Peptides identified with high and median probabilities are shown in green and blue, respectively, and the phosphorylated residues are shown with black asterisks. (C) Identified VP30 phosphopeptides. (D to F) MS/MS spectra of representative phosphorylated peptides from virion-derived MARV VP30: MQQPRHRsR, ARPPsTFNLSKPPPPPK, and SPQDcGsPSLSK, as indicated. Lowercase letters indicate phosphorylation (s) or alkylation (c). Color peaks indicate matched MS/MS fragments, whereby green indicates precursors, and blue and red indicate y and b ions, respectively.

FIG 3

Comparison of MARV VP30 (GenBank accession number P35258) with Ravn virus VP30 (RAVV) (Q1PDC6), EBOV VP30 (EBOV) (Q05323), human respiratory syncytial virus M2-1 (HRSV) (P04545), and human metapneumovirus M2-1 (HMPV) (Q8QN58) proteins. Multiple-sequence alignment was done using Clustal Omega. The blue box in the top group of sequences indicates a region rich in arginines and lysines unique for filoviruses, which supports the role of this protein as a transcription antitermination factor. The red box in the second group indicates a zinc finger motif, and the blue box in this group indicates another region rich in arginines and lysines, followed by a leucine-rich region which indicates its role as an oligomerization domain. Note that two leucines are also conserved in RSV and HMPV. The common core roughly corresponds to residues 132 to 253 of MARV VP30. The symbols indicate various levels of sequence conservation: asterisk, fully conserved; semicolon, conserved between groups of amino acids with strongly similar properties; period, conserved between groups of amino acids with weak similarity. Dashes indicate gaps in sequence alignment. The phosphorylated residues identified in the current study are shown in brown.

FIG 4

Treatment with phosphatase inhibitors increases phosphorylation of MARV VP30. (A to C) Quantitative analysis of MARV peptide expression using SIEVE, version 2.1. Ion elution profiles are shown in blue for control samples, in red for the cells treated with 1E7-03, and in green for the cells treated with okadaic acid. The AALSLTcAGIR peptide was used for normalization. Triangles indicate the time points at which MS/MS was conducted. Representative data from three independent experiments are shown. (D) Ratios of the ion peaks for ARPPsTFNLSKPPPPPK and SsAESSPTNHIPR peptides in 1E7-03- or okadaic acid-treated cells to that in nontreated cells. Values are means ± standard deviations based on three samples. Peaks were integrated, and a P value was calculated using SIEVE, version 2.1. *, P = 8.7 × 10⁻¹⁵; **, P = 4.3 × 10⁻¹¹.

FIG 5

Mapping of the identified phosphorylation sites on the modeled three-dimensional structure of MARV VP30. The modeled full-length structure of MARV VP30 is shown in gold. The molecule comprises the unstructured N-terminal half and the ordered C-terminal half. The indicated phosphorylated residues show oxygen atoms in red. The superimposed structure of the C-terminal half of EBOV VP30 is shown in cyan.

FIG 6

VP30 is required for effective transcription. Luciferase signal in the absence of VP30 was normalized to that in the presence of VP30 in Vero-E6 (A) and 293T (B) cells. Top panels show minigenome analysis; bottom panels show Western blot analysis of Renilla luciferase expression. Values are means ± standard errors of the means based on triplicate samples. *, P < 0.05 for comparisons of results to those with the full set of plasmids (one-way analysis of variance and Dunnett's test). The experiments in panels A and B were performed three times, resulting in essentially similar data.

FIG 7

VP30 dephosphorylation increases MARV transcription. At top, effects of alanine substitutions for Thr and Ser phosphorylation sites on minigenome activity are shown. Luciferase signals of mutated minigenomes were normalized to the signal of nonmutated minigenome (100%). Bottom panels show expression of the mutated VP30 constructs as analyzed by Western blotting. The VP30 Western blot image was spliced from two different gels run in parallel because of the limited number of samples which could be loaded on one gel and to place the samples in the order consistent with the discussion in the rest of the manuscript. The GAPDH Western blot image was spliced from two different gels run in parallel because of the limited number of samples which could be loaded on one gel. Values are means ± standard errors of the means based on triplicate samples. *, P < 0.05 for comparisons of results to those with the nonmutated (wild-type [WT]) VP30 (one-way analysis of variance and Dunnett's test). The experiment was performed at least three times, resulting in essentially similar data.

FIG 8

VP30 phosphorylation inhibits transcription. The effects of alanine and aspartic acid single (A) and cluster (B and C) substitutions of phosphorylated serine residues in VP30 on minigenome activity normalized to activity with nonmutated VP30 (100%) are shown in the graphs. For the experiments shown in panels A and B, a single VP30 concentration of 25 ng was used, whereas for the experiment shown in panel C, three different concentrations of VP30 (25, 50 and 75 ng) were used. Expression of the mutated VP30 constructs was analyzed by Western blotting (A and B, bottom panels). Values are means ± standard errors of the means based on triplicate samples. *, P < 0.05 for comparisons of results to those with the nonmutated (wild-type [WT]) VP30 (one-way analysis of variance and Dunnett's test). The experiments were performed three times, resulting in essentially similar data.

FIG 9

VP30 interacts with NP and VP35. Coimmunoprecipitation was performed of Flag-VP30 phosphorylation mutants with NP (A and B) or HA-VP35 (C and D). 293T cells expressing Flag-tagged VP30 constructs and NP or HA-tagged VP35 were lysed at 48 h posttransfection, and protein complexes were immunoprecipitated (IP) overnight at 4°C using mouse anti-Flag affinity gel or mouse anti-HA agarose. An aliquot for expression control (whole-cell lysate [WCL]) was collected from the cell lysate before precipitation. Western blot analysis performed with anti-NP and anti-Flag antibodies (A, green). (C) Western blotting was performed with anti-HA (green) and anti-Flag (red) antibodies visualized with a LiCor Odyssey Imaging System. Panels B and D show the quantification of immunoprecipitated Flag-VP30 and NP and of Flag-VP30 and HA-VP35, respectively, performed in Image Studio Lite software. Precipitation of VP30 WT by NP/HA-VP35 was set to 100%. Values are means ± standard errors of the means based on duplicate samples. *, P < 0.05 for comparisons of results to those with the nonmutated (wild-type [WT]) VP30 (one-way analysis of variance and Dunnett's test). The experiments were performed at least two times, resulting in essentially similar data.

FIG 10

Treatment with 1E7-03 reduces transcription by promoting binding of phosphorylated VP30 with NP and VP35. (A) Effects of 1E7-03 treatment on minigenome activity. The luciferase signal was normalized to activity in nontreated cells (100%). (B to E) Coimmunoprecipitation of Flag VP30 WT with NP or HA-VP35, as indicated. Western blot analysis with anti-NP and anti-Flag antibodies (green) (B) and with anti-HA (green) or anti-Flag (red) antibodies (D) was visualized with a Li-Cor Odyssey imaging system. Quantification of immunoprecipitated Flag-VP30 and NP and Flag-VP30 and HA-VP35 from the experiments shown in panels B and D, respectively, was performed in Image Studio Lite software. Precipitation of VP30 WT by NP/HA-VP35 was set to 100%. Values are means ± standard errors of the means based on duplicate samples. *, P < 0.05 for results compared to those with the DMSO control (one-way analysis of variance and Dunnett's test). The experiment shown in panel A was performed three times, and the experiments shown in panels B to E were performed at least two times, resulting in essentially similar data.

FIG 11

1E7-03 inhibits replication of MARV. Vero-E6 cells were infected with MARV at an MOI of 0.01 PFU/cell and treated daily with 1E7-03 at 3 μM starting at 24 h prior to the infection or during the infection. (A) Bright-field microscopy of cell monolayers infected with MARV and treated with 1E7-03 starting at the indicated time points. Monolayers on days 1 to 4 are not shown due to the lack of visible cytopathic effect. (B) MARV titers in supernatants of infected monolayers treated with 1E7-03 starting at the indicated time points. Values are means ± standard deviations based on triplicate samples. *, P < 0.05 for results compared to those in the untreated cells (Student's t test). The dotted line indicates the limit of detection of the assay.

FIG 12

A putative model of the role of VP30 phosphorylation in MARV transcription. The phosphorylated form of VP30 (red, left) binds to NP and VP35 and suppresses transcription. The dephosphorylated form of VP30 (green, right) is dissociated from NP and VP35 and does not inhibit transcription. The phosphorylated form of VP30 may work as a repressor of MARV polymerase by associating with NP and VP35. Treatment of cells with 1E7-03 prevents dephosphorylation of VP30 by PP1 that results in suppression of MARV transcription.