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. 2020 Jun 16;94(13):e00115-20.
doi: 10.1128/JVI.00115-20. Print 2020 Jun 16.

Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

Louis-Marie Bloyet #  1 , Benjamin Morin #  1 , Vesna Brusic  1 , Erica Gardner  1 , Robin A Ross  1 , Tegy Vadakkan  2 , Tomas Kirchhausen  2 , Sean P J Whelan  3   4
Affiliations

Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

Louis-Marie Bloyet et al. J Virol. .

Abstract

Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation.IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

Keywords: genome replication; oligomerization; phosphoprotein; polymerase; vesicular stomatitis virus.

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Figures

FIG 1
FIG 1
Characterization of PΔOD. (A) Schematic of VSV PWT and PΔOD showing their modular organization with the N-terminal domain (NTD), L-binding domains (LBD), oligomerization domain (OD), and C-terminal domain (CTD) represented as rectangles. (B) Analysis of purified PWT and PΔOD proteins by polyacrylamide gel electrophoresis on a denaturing gel (left) and a native gel (right). Sizes of two bands of the protein ladder (m) are indicated. (C) SEC-MALS analysis of purified PWT (solid lines) and PΔOD (dashed lines) proteins. The horizontal red traces show the inferred molecular mass. Predicted molecular masses for monomeric and dimeric PWT are 30.3 kDa and 60.6 kDa, respectively. For monomeric and dimeric PΔOD, predicted molecular masses are 22.4 kDa and 44.8 kDa, respectively. Observed experimental molecular masses are 56.2 kDa and 22.6 kDa for PWT and PΔOD, respectively.
FIG 2
FIG 2
Functional analysis of PΔOD in vitro. Reactions were performed in the absence (Ø) or presence of equimolar amounts of PWT or PΔOD using purified L and either a synthetic, naked 19-nt RNA template corresponding to the 3′ leader sequence of the VSV genome (A) or a purified, encapsidated N-RNA template (B). Radioactive products were analyzed by gel electrophoresis on a 20% acrylamide gel (A) or a 1.75% acid-agarose gel containing 6 M urea (B). (A) n = 1 replicate; (B) representative experiment (n = 3 replicates).
FIG 3
FIG 3
Characterization of a recombinant VSV expressing PΔOD. (A) Schematic representation of the recombinant virus genomes. (B) Viral spreading as seen by plaque assay on Vero cells infected with VSV-eGFP-PWT or VSV-eGFP-PΔOD. (C) Viral growth kinetic on Vero cells infected with VSV-eGFP-PWT (PWT, black line) or VSV-eGFP-PΔOD (PΔOD, dotted line) at an MOI of 3. Supernatants were harvested and titers determined at 4, 6, 8, 10, 12, and 24 h postinfection. Statistical analysis was performed by a paired t test. * P, < 0.05; ** P, < 0.005; *** P, < 0.0005; ns, nonsignificant. (D) Analysis of virion protein content. Gradient-purified virions (108 PFU) were denaturated by SDS and heat and analyzed by SDS-PAGE and Coomassie staining. (E) In phosphate-free media supplemented with radioactive [32P]orthophosphate, BSR-T7 cells were treated with 10 μg/ml actinomycin D and 100 μg/ml cycloheximide and infected at an MOI of 100 with VSV-eGFP-PWT or VSV-eGFP-PΔOD. RNA was extracted at 2, 3, 4, 5, and 6 h postinfection and analyzed on a 1.75% acid-agarose gel containing 6 M urea. Representative experiment (n = 4).
FIG 4
FIG 4
FRAP analysis of replication compartments. (A) Schematic representation of the recombinant virus genomes. (B, C) Vero cells were infected at an MOI of 3 with VSV-eGFP/PWT or VSV-eGFP/PΔOD, and eGFP was visualized with a spinning disk confocal microscope at 6 and 10 h postinfection for VSV-eGFP/PWT and VSV-eGFP/PΔOD, respectively. Fluorescence recovery after photobleaching (FRAP) experiments were performed on areas of 4 μm2 located inside compartments. Recovery fluorescence was measured every 500 ms for 50 s. (B) Infected cells before photobleaching (left), and zoomed-in pictures taken at indicated times after photobleaching (right). Dashed and dotted lines delimit the cells and the nucleus, respectively. Squares represent the zoomed-in sections. (C) FRAP data were corrected for background, normalized to the minimum and maximum intensities. The mean is shown on the black line, with the gray zone representing the SD. Mean experimental curves were fitted with double-exponential models (red line; VSV-eGFP/PWT, R2 = 0.997; VSV-eGFP/PΔOD, R2 = 0.996). Statistical comparison of the two data sets was performed using the Kolmogorov-Smirnov test. P < 0.0001.
FIG 5
FIG 5
Effect of PΔOD on viral RNA synthesis. (A) BSR-T7 cells were infected with DI-T for 1 h and transfected with plasmids coding for L, N, and PWT or PΔOD. At 1.5, 2, 2.5, 3, 3.5, or 4 h posttransfection, cells were incubated for 3 h in phosphate-free media supplemented with radioactive [32P]orthophosphate and 10 μg/ml actinomycin D. RNA was harvested and analyzed on a 1.75% agarose gel containing 6 M urea (left). DI-T band intensities were quantified and plotted as percentage of maximal intensity (right). Times of harvest posttransfection are indicated. DI-T bands are marked with an asterisk. Representative experiment (n = 4). (B) BSR-T7 cells were infected with DI-T for 1 h and transfected with plasmids coding for L, N, and PWT or PΔOD. Increasing amounts of plasmid coding for N were transfected with 0.4, 1, 1.6, 2.2, 2.8, and 3.4 μg for PWT and 1, 1.6, 2.2, 2.8, 3.4, and 4 μg for PΔOD. Five hours posttransfection, cells were incubated for 3 h in phosphate-free media supplemented with radioactive [32P]orthophosphate and 10 μg/ml actinomycin D. RNA was harvested and analyzed on a 1.75% agarose gel containing 6 M urea (left). DI-T band intensities were quantified and plotted as percentage of maximal intensity (right). Representative experiment (n = 2).
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