New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes

<div><p>The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.</p></div

G3BP-nsP3 complexes bind viral G RNA in CHIKV-infected cells.

<p>(A) Colocalization of CHIKV nsP3-Cherry (pseudocolored in green) fusion proteins with CHIKV G RNA. Images are presented as MIP of 6 x-y sections (1 μm) through the middle plane of nucleus. Bars: 10 and 3 μm. (B) Colocalization of CHIKV nsP3 with G3BP2, stained with specific Abs at the plasma membrane at 2 h PI. Bars: 10 μm and 3 μm. (C) At 2 h PI, G3BP2 and dsRNA form stripes at the plasma membrane in CHIKV-infected cells. Staining was performed with specific Abs. (D) CHIKV G RNA and G3BP1-GFP strongly colocalize at the plasma membrane in the infected cells. The dsRNA-containing vRCs are formed in close proximity to G3BP-GFP/G RNA complexes and often overlap. Bars: 2 μm. Images in B-D are presented as MIP of x-y sections (1 μm) at the plasma membrane. Yellow arrowheads indicate pre-vRCs, which appear as dsRNA-free G3BP2-positive spots (red) in (C) or G RNA/G3BP1-GFP spots (yellow) in (D). Turquoise arrowheads indicate fully formed, dsRNA-positive vRCs, which contain low levels of G3BP1-GFP. Pink arrowheads indicate pre-vRCs, which contain a dsRNA signal of low intensity and volume and, likely, represent vRCs in the process of synthesis of dsRNA intermediate.</p

Model of FXR- and G3BP-mediated alphavirus replication complex formation.

<p>Binding of FXRs or G3BPs to the PM-bound P123 and P123+nsP4 complexes promotes their interaction with newly synthesized G RNAs and formation of pre-vRCs. In the absence of FXRs or G3BPs, G RNAs are acquired by P123+nsP4 complexes less efficiently and are likely degraded.</p

Repeating aa sequences in VEEV and CHIKV HVDs determine virus replication.

<p>(A) The schematic presentation of the recombinant VEEV genome and modifications introduced into nsP3 HVD sequence. Replacement of repeating sequences in VEEV HVD by those derived from CHIKV HVD makes VEEV/chikvR/GFP replication dependent on G3BP. VEEV/mutHVD/GFP titers were determined at 7 h PI, and VEEV/chikvR/GFP titers were determined at 8 h PI (MOI 0.01). (B) The schematic presentation of the recombinant CHIKV genome and modifications introduced into nsP3 HVD sequence. SINV-specific sequence in CHIKV HVD, but not VEEV repeating element, can support CHIKV replication. Virus titers were determined at 24 h PI (MOI of 0.05). Data in (A) and (B) are presented as mean±SD of 3 biological repeats. NV indicates that the designed mutants were not viable. (C) Colocalization of wt and mutant VEEV nsP3 proteins with FXR1 and G3BP2. Images are presented as MIP of x-y 1-μm-thick sections at the plasma membrane. Pearson's colocalization coefficients are shown in overlay panel (mean±SD, n>6). Bars: 2 μm</p

HVD of VEEV nsP3 binds proteins of FXR family.

<p>(A) Schematic presentation of replicons expressing GFP-HVDveev and GFP-HVDsinv fusion proteins. (B) List of proteins co-isolated with GFP-HVDveev and GFP-HVDsinv from BHK-21 cells. Total spectra for 2 independent immunoprecipitation (IP) experiments are presented. (C) Colocalization of VEEV nsP3 with FXR proteins at 6 h PI at an MOI of 20. Pearson's colocalization coefficients are shown in first overlay panels (mean±SD, n cells >12). Scale bars: 20 μm for 2 left panels or 3 μm for 3 right panels.</p

Any single FXR or G3BP protein supports virus replication.

<p>(A, B) A single, ectopically expressed FXR or G3BP protein supports replication of VEEV and CHIKV, respectively. Single FXR or G3BP-GFP proteins were stably expressed in <i>Fxr</i> tKO or <i>G3bp</i> dKO cells, respectively. VEEV titers were determined at 8 h PI at an MOI 0.05. CHIKV titers were determined at 7 h PI at an MOI of 0.1. (C, D) Analysis of function of FXR1 and G3BP1 domain deletion mutants in VEEV and CHIKV replication, respectively. The experiments were performed in stable cell lines, established in <i>Fxr</i> tKO or <i>G3bp</i> dKO cells. VEEV titers were assessed at 8 h PI (MOI 0.05), and CHIKV titers were determined at 7 h PI (MOI 0.1). n.d. indicates that virus replication was below the detection limit. n.a. indicates that this cell line was not available. Data are mean±SD of 3 biological repeats.</p

The knockout of <i>Fxr</i> or <i>G3bp</i> genes differentially affects replication of alphaviruses.

<p>(A) Left panels: growth curves of the indicated alphaviruses in NIH 3T3, <i>Fxr</i> dKO and <i>Fxr</i> tKO cells (MOI 1). Right panels: bar graphs show titers of the indicated viruses in NIH 3T3 and <i>Fxr</i> tKO cells at 8 h PI (MOI 0.05, three biological repeats). (B) Left panels: growth curves of the indicated alphaviruses in NIH 3T3, <i>G3bp2</i> KO and <i>G3bp</i> dKO cells (MOI 0.01). Right panels: bar graphs show titers of the indicated viruses in NIH 3T3 and <i>G3bp</i> dKO cells at 8 h PI (MOI 0.05, three biological repeats). (C, D) Sizes of plaques developed in NIH 3T3/<i>Fxr</i> tKO (C) or NIH 3T3/<i>G3bp</i> dKO (D) cells by the indicated viruses. Due to strong reduction in infectivity of KO cells wells with different virus dilutions are shown. Data in bar graphs shown in (A) and (B) are mean±SD of 3 biological repeats. DL indicates a detection limit.</p

FXRs and G3BPs facilitate vRC formation and RNA replication.

<p>(A) FACS analysis of NIH 3T3, <i>Fxr</i> tKO and <i>G3bp</i> dKO cells, infected with VEEV/GFP and CHIKV/GFP (4 h PI, MOI 10). (B) Replication rates of a chimeric virus VEE/CHIKV in the indicated cells (MOI of 0.1). (C) Translation of nLuc-encoding RNA with alphavirus genome-specific 5’UTRs in the indicated cell lines. Data are mean±SD of 3 biological repeats. Deviations are too small to be visible in the used log scale. (D) Accumulation of VEEV- and CHIKV-specific G RNAs, measured by RT-qPCR, in the indicated cells infected at an MOI of 10 (mean±SD, 3 repeats). (E) Accumulation of nsP3 and capsid protein in <i>Fxr</i> dKO, <i>Fxr</i> tKO cells and parental NIH 3T3 cells. Cells were infected with VEEV TC-83 at an MOI of 20, harvested at different times PI, and the lysates were analyzed by Western blot using nsP3-specific and capsid-specific Abs. Quantitative analysis was performed on LI-COR imager. n.d. indicates that protein concentration was below the detection level. (F) Numbers of vRCs per cell at different times PI with VEEV TC-83. The indicated <i>p</i> values are between NIH 3T3 and KO cells at the same time points. (G) Numbers of vRCs in indicated cells at 3 h PI with VEEV TC-83. The p values in black are between NIH 3T3 and KI cell lines, the p values in blue are between <i>Fxr</i> tKO and KI cells. Data in F and G are presented as median with interquartile range. The <i>p</i> values were estimated using Mann-Whitney test; n cells per group > 29 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005810#sec010" target="_blank">Materials and Methods</a> for details).</p

Levels of resistance of alphavirus RNA translation to IFN-β is determined by 5’UTRs of their genomes.

<p><b>(A)</b> The schematic representation of EIL/5’nLuc/VEEV genomes with different 5’UTRs in the nLuc-encoding SG RNAs and the computer predictions (m-Fold) of the RNA foldings. <b>(B)</b> The schematic representation of the EIL/5’EIL-IRES-nLuc/VEEV genome, containing EMCV IRES downstream of the EILV SG RNA-specific 5’UTR. Because of the IRES presence, nLuc is likely translated from both viral genome and SG RNA. <b>(C)</b> wt MEFs were treated for 20 h with different concentrations of IFN-β or remained mock-treated and then infected with the indicated viruses for 1 h at 37°C. The MOI used was adjusted for each virus to obtain similar nLuc activity in the mock-treated samples. After infection, cells were incubated in complete medium at 37°C, and nLuc activity was assessed at 4 h post infection. The results were normalized to the nLuc activity in the samples of mock-treated, infected cells. The experiments were repeated multiple times with reproducible results. Panels represent the data obtained in a single experiment. All of the measurements were performed in triplicates, and standard deviations are too small to be seen.</p

Alphavirus resistance to IFIT1 can be manipulated genetically and leads to virus attenuation.

<p><b>(A)</b> The computer prediction of the 5’UTRs of the parental VEEV TC-83 and 5’mutVEEV viruses. To analyze the replication rates, wt MEFs and IFIT1 KI/1 cells were infected at an MOI of 5 PFU/cell with VEEV TC-83 and 5’mutVEEV, media were replaced at the indicated times PI, and virus titers were determined on BHK-21 cells. <b>(B)</b> At 48 h post infection, cells used for analysis of virus replication were stained with crystal violet to assess for CPE. <b>(C)</b> C7/10 cells were infected at an MOI of 10 PFU/cell with VEEV TC-83 and 5’mutVEEV, media were replaced at the indicated times PI, and virus titers were determined on BHK-21 cells. <b>(D)</b> The schematic representation of the CHIKV/GFP 181/25 genome and computer predictions of the original and newly designed 5’UTRs. Rescued viruses were used to infect wt MEFs, IFIT1 KI/2 and IFIT1 KI/3 cell lines at an MOI of 5 PFU/cell, and at 24 h post infection, virus titers were determined by plaque assay on BHK-21 cells. <b>(E and F)</b> Six-day-old NIH Swiss mice were infected via s.c. route with the indicated doses of VEEV TC-83 or the designed VEEV variant with mutated 5’UTR (5’mutVEEV). Mice were monitored daily in terms of weight change <b>(E)</b> and survival <b>(F)</b>.Unpaired T-test was used to compare weight change between the VEEV TC-83 and 5’mutVEEV groups (both 2x10⁵ and 10⁶ PFU) at day 4 and 5 post-infection. ***: p<0.001. Survival curves of the VEEV TC-83 and 5’mutVEEV groups (both 2x10⁵ and 10⁶ PFU) are significantly different (p-values <0.001).</p