Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

Abstract

The four brome mosaic virus (BMV) RNAs (RNA1 to RNA4) are encapsidated in three distinct virions that have different disassembly rates in infection. The mechanism for the differential release of BMV RNAs from virions is unknown, since 180 copies of the same coat protein (CP) encapsidate each of the BMV genomic RNAs. Using mass spectrometry, we found that the BMV CP contains a complex pattern of posttranslational modifications. Treatment with phosphatase was found to not significantly affect the stability of the virions containing RNA1 but significantly impacted the stability of the virions that encapsidated BMV RNA2 and RNA3/4. Cryo-electron microscopy reconstruction revealed dramatic structural changes in the capsid and the encapsidated RNA. A phosphomimetic mutation in the flexible N-terminal arm of the CP increased BMV RNA replication and virion production. The degree of phosphorylation modulated the interaction of CP with the encapsidated RNA and the release of three of the BMV RNAs. UV cross-linking and immunoprecipitation methods coupled to high-throughput sequencing experiments showed that phosphorylation of the BMV CP can impact binding to RNAs in the virions, including sequences that contain regulatory motifs for BMV RNA gene expression and replication. Phosphatase-treated virions affected the timing of CP expression and viral RNA replication in plants. The degree of phosphorylation decreased when the plant hosts were grown at an elevated temperature. These results show that phosphorylation of the capsid modulates BMV infection.

Importance: How icosahedral viruses regulate the release of viral RNA into the host is not well understood. The selective release of viral RNA can regulate the timing of replication and gene expression. Brome mosaic virus (BMV) is an RNA virus, and its three genomic RNAs are encapsidated in separate virions. Through proteomic, structural, and biochemical analyses, this work shows that posttranslational modifications, specifically, phosphorylation, on the capsid protein regulate the capsid-RNA interaction and the stability of the virions and affect viral gene expression. Mutational analysis confirmed that changes in modification affected virion stability and the timing of viral infection. The mechanism for modification of the virion has striking parallels to the mechanism of regulation of chromatin packaging by nucleosomes.

FIG 1

Mass measurements of BMV CP. (A) Genetic map showing locations of BMV CP residues predicted to be phosphorylated, acetylated (Ac), and methylated. Residues predicted to be phosphorylated (S,T) are above the bar, and residues predicted to be acetylated or methylated (K) are below the bar. The location of the CP residues that form the globular region is shaded in gray. (B and C) MS was performed on the B1 (B) and B2.3/4 (C) virions to examine BMV PTMs (solid black line). Peak A corresponds to the BMV CP with the first methionine cleaved and an N-terminal acetyl group. Peak B corresponds to the putatively phosphorylated CP. CIP was used to confirm the presence of CP phosphorylation (dashed line). The abundance of the modified CP was calculated by integrating the areas encompassed by the peaks. The abundance of peak A was normalized to 100% to allow comparison to the other forms.

FIG 2

BMV capsid modification analysis. (A) Modifications of the BMV capsid observed using MS/MS. Numbers show the relative abundance of the modified peptides compared to that of the unmodified peptide in the same sample. Phos., phosphorylation; N-term, N terminus. (B) MS/MS spectra showing methylation of K41 with the b and y ions labeled. (C) MS/MS spectra of acetylated K64 with the b and y ions labeled. (D) MS/MS spectra of phosphorylated T62 with the b and y ions, as well as those observed with neutral loss, labeled. (E) MS spectra of the partially digested N-terminal 10 residues, confirming phosphorylation of this region. Ace, acetyl. (F) Abundance of phosphorylated residues compared to that of the unmodified peptides after phosphopeptide enrichment.

FIG 3

Phosphorylation of the BMV capsid affects virion stability. (A) DSF profile of B1 and B2.3/4 virions mock treated and CIP treated. -R′(T), rate of change of fluorescence. (B) Protein kinase C treatment alters the DSF profiles of BMV virions. B1 and B2.3/4 virions were mock treated or treated with protein kinase C. (C) Resistance to protease, measured by determination of the amount of intact CP, of B1 and B2.3/4 virions mock treated and CIP treated. The vertical bars show the range for 1 standard deviation.

FIG 4

The phosphorylation state of the BMV CP can alter the conformation of the virions. (A and B) Electron micrographs of ice-embedded B1 virions (A) and B2.3/4 virions (B) mock treated and CIP treated. (Insets) Translationally aligned averaged images showing two concentric rings of the capsid and the capsid-RNA. Bar, 50 nm. (C) One-dimensional radial density profiles of the 3D reconstructions of B1 and B2.3/4 virions either mock treated or CIP treated. (D to G) Central slices (D and E) and bottom-half surface-shaded interiors (F and G) of the 3D reconstructions of mock-treated B1 virions (D and F, left), CIP-treated B1 virions (D and F, right), mock-treated B2.3/4 virions (E and G, left), and CIP-treated B2.3/4 virions (E and G, right). (H and I) Overlays of 3D maps of mock-treated B1 and B2.3/4 virions (H, left), mock- and CIP-treated B1 virions (H, right), mock- and CIP-treated B2.3/4 virions (I, left), and CIP-treated B1 and B2.3/4 virions (I, right).

FIG 5

Internal density of the reconstructed BMV virions. (A) Resolution estimation of the cryo-EM 3D maps of BMV particles. The final resolutions for mock-treated B1, CIP-treated B1, mock-treated B2.3/4, and CIP-treated B2.3/4 virions are 7.6 Å, 8.9 Å, 6.7 Å, and 8.1 Å, respectively. The resolution was estimated by use of the Fourier shell correlation (FSC), determined at a 0.5 cutoff. (B) Fitting of atomic model into cryo-EM density maps of each BMV particle. (C) Difference map of the internal density. The difference map was calculated by subtracting the low-pass filtered X-ray model (using the rigid body fitting result) from each cryo-EM density map, and the substantial difference was rendered at the contour level equivalent to the original cryo-EM structure (gray) or at the high-contour level (color). (Top) Bottom-half view of internal density of each BMV particle; (middle) front-half view of the difference map shown in different contour levels (gray, low; color, high); (bottom) high front-half view of difference map rendered at the high contour level to show the dense structural features.

FIG 6

Phosphorylation of the BMV capsid affects the capsid-RNA interaction. (A to C) The number of RNA sequences contacting the capsid within mock-treated and CIP-treated virions identified by CLIP-seq analysis. Normalized coverage is shown. The identities of the majority of sequences that contact the capsid do not change as a function of capsid phosphorylation, but differences in intensity were observed. Peaks with an asterisk represent those with a greater than 20% change following CIP treatment. MP, movement protein; SLC, stem loop C. (D) Previously characterized BMV RNA regulatory elements that had at least a 10% change in expression following CIP treatment. The nucleotides column displays the locations of the regulatory elements. The final column shows the percent change following CIP treatment, and the nucleotide position where the change was observed is given in parentheses. PE, packaging element; IRE, intergenic replication enhancer; Rep., replication. (E) Resistance to RNase A digestion, as measured by the abundance of intact RNA derived from B1 and B2.3/4 virions before and after CIP treatment, followed by RNase treatment. Black, CIP-treated samples; gray, mock-treated samples. The range for 1 standard deviation is shown. ns, no significant difference; ***, P < 0.005.

FIG 7

Phosphorylation affects BMV replication and virion production. (A) BMV CP accumulation in wheat seedlings inoculated for 72 and 102 h with mock- or CIP-treated BMV virions. Each sample was from a separately inoculated pot of wheat seedlings. Total wheat seedlings were harvested and processed for analysis of BMV CP accumulation by Western blotting. The CP was detected with a polyclonal antiserum that recognizes the BMV CP. BMV was undetectable at 24 and 48 h postinoculation, indicating that the signal is from CP accumulation during infection. LC, loading control. Numbers under the blots indicate quantified CP compared to LC. (B) (−)RNA1 (R1) and (−)RNA2 (R2) accumulation at 16, 18, and 20 h postinoculation in plants infected with mock-treated or CIP-treated BMV. The range for 1 standard deviation is shown at each time point.

FIG 8

A phosphomimetic in the N-terminal arm of the BMV CP affects BMV infection and RNA encapsidation. (A) MS spectra of the T6E mutant. Higher levels of phosphorylation were observed for CP with the T6E mutation than WT BMV CP, as indicated by the +80 above the peak. (B) MS spectra of CP with the T6A mutation. Significantly higher levels of phosphorylation were not observed in the CP T6A mutant than WT BMV CP, as indicated by the +80 above the peak. (C) Virions with the T6E mutation dissociated more easily than the WT virions, consistent with the result that phosphorylation decreases virion stability. (D) DSF profile of virions with the T6E mutation mock treated or CIP treated. The high levels of phosphorylation significantly decrease the stability of the virions. (E) Abundance of BMV CP in wheat seedlings inoculated for 72 and 102 h with virions with the T6E mutation or WT BMV virions. Each sample was obtained from a separately inoculated pot of wheat seedlings. CP accumulation was detected by Western blotting. (F) (−)RNA1 and (−)RNA2 accumulation at 20, 22, and 24 h in plants infected with WT or T6E mutant BMV virions. The range for 1 standard deviation is shown at each time point. (G) Percentage of encapsidated RNA in the WT (gray bars) and T6E mutant (black bars) virions. The range for 1 standard deviation is shown. *, P < 0.05.

FIG 9

Environmental effects on BMV CP phosphorylation and infection. (A) MS analysis of the B1 and B2.3/4 virions from plants grown at 25°C and 32°C. The abundance of phosphorylated CP decreased in the virions grown at 32°C. (B) DSF profile of the B1 and B2.3/4 virions grown at 25°C and 32°C. B1 particles grown at 32°C had a population with a Tm_app of over 80°C, while the majority of the B2.3/4 particles grown at 32°C had a Tm_app of 69°C. (C) (−)RNA1 and (−)RNA2 at 20 h postinoculation in plants grown at 25°C produced from virions at 25°C (left) and 32°C (right). (D) (−)RNA1 and (−)RNA2 at 20 h postinoculation in plants grown at 32°C produced from virions at 25°C (right) and 32°C (left) (D). The range for 1 standard deviation is shown. ***, P < 0.005.