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. 2022 Sep 28;96(18):e0109322.
doi: 10.1128/jvi.01093-22. Epub 2022 Sep 13.

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling

Chorong Park  1 , Derek Walsh  1
Affiliations

RACK1 Regulates Poxvirus Protein Synthesis Independently of Its Role in Ribosome-Based Stress Signaling

Chorong Park et al. J Virol. .

Abstract

Receptor for activated C kinase 1 (RACK1) is a small ribosomal subunit protein that is phosphorylated by vaccinia virus (VacV) to maximize translation of postreplicative (PR) mRNAs that harbor 5' polyA leaders. However, RACK1 is a multifunctional protein that both controls translation directly and acts as a scaffold for signaling to and from the ribosome. This includes stress signaling that is activated by ribosome-associated quality control (RQC) and ribotoxic stress response (RSR) pathways. As VacV infection activates RQC and stress signaling, whether RACK1 influences viral protein synthesis through its effects on translation, signaling, or both remains unclear. Examining the effects of genetic knockout of RACK1 on the phosphorylation of key mitogenic and stress-related kinases, we reveal that loss of RACK1 specifically blunts the activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at late stages of infection. However, RACK1 was not required for JNK recruitment to ribosomes, and unlike RACK1 knockout, JNK inhibitors had no effect on viral protein synthesis. Moreover, reduced JNK activity during infection in RACK1 knockout cells contrasted with the absolute requirement for RACK1 in RSR-induced JNK phosphorylation. Comparing the effects of RACK1 knockout alongside inhibitors of late stage replication, our data suggest that JNK activation is only indirectly affected by the absence of RACK1 due to reduced viral protein accumulation. Cumulatively, our findings in the context of infection add further support for a model whereby RACK1 plays a specific and direct role in controlling translation of PR viral mRNAs that is independent of its role in ribosome-based stress signaling. IMPORTANCE Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal protein that regulates translation directly and mediates signaling to and from the ribosome. While recent work has shown that RACK1 is phosphorylated by vaccinia virus (VacV) to stimulate translation of postreplicative viral mRNAs, whether RACK1 also contributes to VacV replication through its roles in ribosome-based stress signaling remains unclear. Here, we characterize the role of RACK1 in infected cells. In doing so, we find that RACK1 is essential for stress signal activation by ribotoxic stress responses but not by VacV infection. Moreover, although the loss of RACK1 reduces the level of stress-associated JNK activation in infected cells, this is an indirect consequence of RACK1's specific requirement for the synthesis of postreplicative viral proteins, the accumulation of which determines the level of cellular stress. Our findings reveal both the specific role of RACK1 and the complex downstream effects of its control of viral protein synthesis in the context of infection.

Keywords: RACK1; poxvirus; ribosome; ribosome quality control; ribotoxic stress responses; signaling; stress; stress signaling; translation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Comparison of VacV infection in HAP1 and NHDF cells. HAP1 and normal human dermal fibroblasts (NHDF) cells were infected with VacV Western reserve (WR) at a multiplicity of infection (MOI) of 25 for the indicated period of time. (A) Cells were stained with Hoechst dye and I3 (red) antibody. Representative images are shown for each cell type and time point. (B) Cells were labeled with 35S-Met/Cys for 30 min prior to harvesting at the indicated times postinfection. The samples were subjected to polyacrylamide gel analysis and autoradiography to detect nascent protein synthesis (top) or Western blotting for the indicated viral proteins (bottom). The results are representative of at least three independent experiments. hpi, hours postinfection.
FIG 2
FIG 2
VacV infection activates mitogenic and stress-related mitogen-activated protein kinase (MAPK) signaling pathways at postreplicative stages of infection. (A) HAP1 cells were infected with VacV at MOI 25 and harvested at 0, 6, and 24 h hpi. The samples were analyzed by Western blotting with the indicated antibodies. (B) NHDF, HEK293A, and MRC5 cells were infected and analyzed as described for HAP1 cells for panel A. The results are representative of at least three independent experiments. JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase.
FIG 3
FIG 3
Loss of RACK1 specifically reduces postreplicative viral protein production. HAP1 parental (par.) or two HAP1 RACK1 knockout cell lines (KO#1, KO#2) were infected with VacV at MOI 25 for 24 h in the presence or absence of AraC (40 μg/mL). (A, B) Total DNA (A) or RNA (B) were extracted and measured by RT-PCR with using I3 (early) and A14 (late) gene primers. The results are representative of at least two independent experiments. (C) The cells were lysed and subjected to Western blotting with the indicated antibodies. The results are representative of at least three independent experiments.
FIG 4
FIG 4
VacV-induced activation of JNK is impaired in RACK1 knockout cells. (A) HAP1 parental (par.) or two HAP1 RACK1 knockout cell lines (KO#1, KO#2) were infected with VacV at MOI 25 and harvested at the indicated time points. The cells were lysed and subjected to Western blotting with the indicated antibodies. (B) HAP1 RACK1 knockout (KO#1) cells expressing either FLAG-control or FLAG-tagged RACK1 were infected as described for panel A. The cells were lysed and subjected to Western blotting with the indicated antibodies. The results are representative of at least three independent experiments.
FIG 5
FIG 5
VacV replication is required for maximal JNK and p38 activation. HAP1, NHDFs, HEK 293A, and MRC5 cells were infected with VacV at MOI 25 for 24 h in the presence or absence of phosphonoacetic acid (PAA) (400 μg/mL) (A) and AraC (40 μg/mL) (B). The cells were lysed and subjected to Western blotting with the indicated antibodies. The results are representative of two independent experiments for each inhibitor and cell type.
FIG 6
FIG 6
RACK1 does not regulate JNK recruitment to ribosomes. HAP1 parental or HAP1 RACK1 knockout (KO#1) cells were infected with VacV at MOI 25 and harvested for polysome fractionation. Fractions were trichloroacetic acid (TCA) precipitated and subjected to Western blotting with the indicated antibodies. A sample of total lysate (L) is shown alongside each fraction. Fractions are labeled to illustrate the distribution of initiation complexes (eIF4F and eIF4F+eIF3) followed by 43S (40S subunit bound by eIFs), 60S, and assembled 80S ribosomes (distributed as 80S monosomes, disomes, and polysomes). Representative small (RPS2, RACK1) and large subunit proteins (RPL11, RPL13a) are shown. eIF, eukaryotic initiation factor; SSU, small subunit; LSU, large subunit. The results are representative of three independent experiments.
FIG 7
FIG 7
Ribosome profiles are altered at postreplicative stages of vaccinia virus (VacV) infection. Mock or VacV-infected HAP1 parental and RACK1 KO cells were lysed in polysome buffer and subjected to sucrose gradient centrifugation. Polysome traces were measured by detecting RNA at 254 nm. (A) Polysome profiles in uninfected cells. Note that the absence of RACK1 only modestly reduces the level of polysomes, in particular larger polysomes. (B) Polysome profiles at early stages of the VacV infection (6 hpi) are very similar to those of uninfected cells and exhibit similar effects of RACK1 loss as in panel A. (C) Polysome profiles at postreplicative stages of the VacV infection (24 hpi) illustrating the reduction in heavy polysomes, the increase in 80S monosomes, and the appearance of disome peaks, indicated and marked with red circles. Loss of RACK1 at this stage of infection causes a notable loss of polysomes and the disome shoulder. The results are representative of three independent experiments.
FIG 8
FIG 8
Differential requirements for RACK1 in JNK activation during poxvirus infection versus ribotoxic stress responses. (A) HAP1 parental and RACK1 KO cells were treated with anisomycin (ANS) (10 μM) for 1 h or infected with VacV at MOI 25 for 24 h. The cells were lysed and subjected to Western blotting with the indicated antibodies. ANS-induced JNK activation is completely dependent on RACK1, while VacV-induced JNK activation is not. (B) Validation of JNK inhibitor VIII efficacy in cells treated with ANS or infected with VacV. JNKVIII blocks JNK activity but not its phosphorylation. To verify inhibitory efficacy, HAP1 cells were treated with dimethyl sulfoxide (DMSO) solvent control or ANS (10 μM) for 1 h or infected with VacV at MOI 25 for 24 h in the presence of JNK inhibitor VIII at the indicated concentrations. Uninfected or infected HAP1 cells were treated with DMSO as a solvent control. Phosphorylated c-Jun was used as a readout for inhibition of JNK activity. (C) JNK activity is not required for VacV protein synthesis. HAP1 cells were infected with VacV as described for panel B. Representative early-intermediate (I3) and late (F17, A14) VacV proteins were analyzed by Western blotting. Early and late viral protein levels were unaffected by JNK inhibition. The results are representative of three independent experiments.
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