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. 2010 Jul;84(13):6720-32.
doi: 10.1128/JVI.01983-09. Epub 2010 Apr 14.

Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus replication

Ileana M Cristea  1 , Heather Rozjabek, Kelly R Molloy, Sophiya Karki, Laura L White, Charles M Rice, Michael P Rout, Brian T Chait, Margaret R MacDonald
Affiliations

Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus replication

Ileana M Cristea et al. J Virol. 2010 Jul.

Abstract

Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.

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Figures

FIG. 1.
FIG. 1.
Characteristics of viruses used in the present study. (A) Schematic diagrams of the viruses utilized in this study are shown. TE/5′2J expresses two subgenomic (SG) RNAs, indicated by arrows above the diagram; the 5′ SG RNA express foreign genes inserted using restriction sites (black box) between the two SG transcriptional start sites, while the 3′ SG RNA encodes the viral structural proteins. An indicates the poly(A) tail. Three alanine residues expressed by SINV TE/nsP3-3A are indicated at the carboxyl terminus of nsP4, the viral RNA-dependent RNA polymerase. The location of the 3×Flag tag at the carboxyl termini of nsP4 and GFP in SINV TE/nsP4-3×Flag and TE/5′2J/GFP-3×Flag, respectively, is indicated. (B) The RNA sequences of the parental (TE/5′2J) and derived viruses are shown, with the amino acid sequence shown above. An asterisk indicates a stop codon. The arrow indicates the SG RNA derived from the 5′ SG promoter. Nucleotides mutated to disrupt SG promoter function are underlined. (C) Comparison of the infectivity of the viral constructs. BHK-21 cells were electroporated with 1 μg of each RNA in duplicate (or triplicate, TE/nsP4-3×Flag), and the number of infectious centers was determined. Mean plaque diameter, as assessed by measurement of 30 to 40 plaques for each virus, is shown, with the standard deviation indicated. The asterisk indicates a statistically different mean diameter from the TE/5′2J parental virus (P = 0.0015, unpaired t test). The remaining electroporated cells were placed in culture and 1 day later the medium overlying the cells was removed and pooled to generate a virus stock. The titer of infectious virus, determined by standard plaque assay, is indicated.
FIG. 2.
FIG. 2.
Identification of nsP4-associated host factors using isolations on magnetic beads and mass spectrometry. Rat2 cells were infected with SINV TE/5′2J/GFP-3×Flag (control) virus expressing Flag-tagged GFP or SINV TE/nsP4-3×Flag (nsP4) virus expressing Flag-tagged nsP4 and at the indicated times after infection the Flag-tagged proteins and associated factors were isolated by affinity purification using magnetic beads conjugated to anti-Flag antibodies as described in Materials and Methods. The isolated proteins were resolved by SDS-PAGE and stained with Coomassie blue, and the proteins present in each entire lane were identified by mass spectrometry. (A) The identity of viral (blue) and prominent host (black) proteins in the nsP4 samples is indicated. Vimentin (indicated with an asterisk) was predominant in both the control and nsP4 samples. GFP, isolated due to the 3×Flag tag present on the GFP-3×Flag, is indicated in the control samples. (B) The identity of the proteins present in the control sample harvested at 6 hpi is indicated, forming a list of likely contaminants for our nsP4 isolations. Table 2 and Table S1 in the supplemental material provide the complete list of identified proteins.
FIG. 3.
FIG. 3.
G3BP1 colocalizes with nsP4 in SINV-infected cells. Rat2 cells were infected with SINV for the indicated times or were mock infected. After fixation the cells were analyzed by indirect immunofluorescence confocal microscopy as described in Materials and Methods. The individual G3BP1 and nsP4 signals are shown (left 2 panels, white), as well as the merged G3BP1 (green) and nsP4 (red) images. Nuclei are shown in blue in the merged images. Bars, 10 μm. A representative fluorescence intensity profile is shown on the right for each sample.
FIG. 4.
FIG. 4.
Silencing of G3BP1 and G3BP2 enhances SINV polyprotein expression. (A) 293T cells were transfected with an irrelevant siRNA (Irr) or siRNA smartpools targeting G3BP1, G3BP2, or both G3BP1 and G3BP2 as indicated. Two days later, the cells were infected for 1 h (MOI of ∼5) with SINV Toto1101/Luc at 37°C (replication competent, open bars) or Toto1101/Luc:ts110 at 40°C (replication incompetent, shaded bars). Fresh medium was added, and the cells were incubated for an additional 5 h at the respective temperatures prior to lysis for determination of luciferase activity. Bars indicate the mean luciferase activity, normalized for cell viability; error bars indicate the standard deviation of triplicate samples. Asterisks indicate values significantly different than the respective control (Irr) for each virus, which was set at 100% (unpaired t test; *, P < 0.05; **, P < 0.01). The results shown are representative of several similar experiments. (B) The temperature sensitive nature of Toto1101/Luc:ts110 in 293T cells was confirmed by metabolic labeling. RNA was harvested from mock-infected or Toto1101/luc:ts110-infected cells (MOI of ∼10) that had been incubated 24 h at permissive (28°C) or nonpermissive (40°C) temperatures in the presence of actinomycin D and [5-3H]uridine. Total RNA (3 μg) was size separated by denaturing agarose electrophoresis, and the newly synthesized RNA was visualized by fluorography. A 23 h (28°C) and 12 days (40°C), exposure of the dried gel was obtained. Below, a reverse image of the ethidium-stained gel obtained prior to fluorography shows rRNA as a loading control. (C) G3BP1 and G3BP2 RNA silencing efficacy was determined in samples treated as in panel A, but harvested prior to infection. The table indicates the G3BP RNA levels relative to levels present in the irrelevant siRNA-treated sample (set at 1.0). GAPDH RNA levels were used for normalization. The data are the mean relative levels and standard deviations from three independent experiments. (D) Western blot analysis confirms reductions in G3BP1 protein levels. Equal volumes of lysates from cells treated as in C with the indicated siRNAs were loaded onto 8% polyacrylamide gels and subjected to Western blot analysis using anti-G3BP1 (top) or anti-β-actin (bottom) antibodies.
FIG. 5.
FIG. 5.
Silencing of the G3BPs has minimal effects on SINV RNA levels. 293T cells were treated with irrelevant siRNA (□) or siRNAs targeting both G3BP1 and G3BP2 (░⃞) using a final siRNA concentration of 80 nM. After 2 days, the cells were infected with Toto1101/Luc (MOI of ∼5). At the indicated times after infection, the cells were washed, and RNA was harvested and utilized for quantification of SINV RNA levels by quantitative real-time RT-PCR. Known copy numbers of in vitro-transcribed SINV RNA were used to generate a standard curve. For each condition, RNA from a single well was used to prepare duplicate cDNA samples, which were assayed in duplicate; bars represent the mean values obtained from the cDNAs ± the standard errors of the means. The asterisk indicates values significantly different than the irrelevant silenced sample at a given time point (unpaired t test, P < 0.05). Similar results were obtained in another independent experiment, although there was no statistically significant difference at the 4-h time point.
FIG. 6.
FIG. 6.
Silencing of the G3BPs has a sustained effect on SINV polyprotein expression and a small enhancing effect on SINV virion production. 293T cells were treated with an irrelevant siRNA (•) or siRNAs targeting G3BP1 and G3BP2 (○) and were infected 2 days later with SINV. (A) Cells seeded in 24-well plates were infected with Toto1101/Luc (MOI of ∼5) and were harvested at the indicated times. The data are the mean luciferase activities relative to the activity of the 0-h irrelevant silenced sample; error bars indicate the standard deviation of triplicate samples. Similar results were obtained in one other independent experiment. (B) Cells seeded in 12-well plates were infected with Toto1101 (MOI of ∼0.01), and the amount of virus released into the medium of duplicate samples was determined by plaque assay titration on BHK-21 cells. Separate wells were utilized for each time point. The data are mean log titers ± the standard errors of the means; error bars are obscured by the symbols. Similar results were obtained in another independent experiment. For both panels A and B, asterisks indicate values significantly different than the irrelevant silenced sample at a given time point (unpaired t test; *, P < 0.05; **, P < 0.01).
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References

    1. Atasheva, S., R. Gorchakov, R. English, I. Frolov, and E. Frolova. 2007. Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J. Virol. 81:5046-5057. - PMC - PubMed
    1. Bick, M. J., J.-W. N. Carroll, G. Gao, S. P. Goff, C. M. Rice, and M. R. MacDonald. 2003. Expression of the zinc-finger antiviral protein inhibits alphavirus replication. J. Virol. 77:11555-11562. - PMC - PubMed
    1. Blethrow, J. D., C. Tang, C. Deng, and A. N. Krutchinsky. 2007. Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes. PLoS One 2:e358. - PMC - PubMed
    1. Chen, G. I., and A. C. Gingras. 2007. Affinity-purification mass spectrometry (AP-MS) of serine/threonine phosphatases. Methods 42:298-305. - PubMed
    1. Costa, M., A. Ochem, A. Staub, and A. Falaschi. 1999. Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway. Nucleic Acids Res. 27:817-821. - PMC - PubMed

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