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Hantaviruses use the endogenous host factor P58IPK to combat the PKR antiviral response

['Zekun Wang', 'Joint National Laboratory For Antibody Drug Engineering', 'School Of Basic Medical Sciences', 'Henan University', 'Kaifeng', 'Songyang Ren', 'Western University Of Health Sciences', 'Pomona', 'California', 'United States Of America']

Date: 2021-11

Hantavirus nucleocapsid protein (NP) inhibits protein kinase R (PKR) dimerization by an unknown mechanism to counteract its antiviral responses during virus infection. Here we demonstrate that NP exploits an endogenous PKR inhibitor P58 IPK to inhibit PKR. The activity of P58 IPK is normally restricted in cells by the formation of an inactive complex with its negative regulator Hsp40. On the other hand, PKR remains associated with the 40S ribosomal subunit, a unique strategic location that facilitates its free access to the downstream target eIF2α. Although both NP and Hsp40 bind to P58 IPK , the binding affinity of NP is much stronger compared to Hsp40. P58 IPK harbors an NP binding site, spanning to N-terminal TPR subdomains I and II. The Hsp40 binding site on P58 IPK was mapped to the TPR subdomain II. The high affinity binding of NP to P58 IPK and the overlap between NP and Hsp40 binding sites releases the P58 IPK from its negative regulator by competitive inhibition. The NP-P58 IPK complex is selectively recruited to the 40S ribosomal subunit by direct interaction between NP and the ribosomal protein S19 (RPS19), a structural component of the 40S ribosomal subunit. NP has distinct binding sites for P58 IPK and RPS19, enabling it to serve as bridge between P58 IPK and the 40S ribosomal subunit. NP mutants deficient in binding to either P58 IPK or RPS19 fail to inhibit PKR, demonstrating that selective engagement of P58 IPK to the 40S ribosomal subunit is required for PKR inhibition. Cells deficient in P58 IPK mount a rapid PKR antiviral response and establish an antiviral state, observed by global translational shutdown and rapid decline in viral load. These studies reveal a novel viral strategy in which NP releases P58 IPK from its negative regulator and selectively engages it on the 40S ribosomal subunit to promptly combat the PKR antiviral responses.

Activation of PKR during virus infection shuts down the host translation machinery and creates an antiviral state to create obstacles for viral protein synthesis. Our results demonstrate that hantaviruses hijack an endogenous PKR inhibitor P58 IPK to combat the PKR antiviral response. The PKR remains associated with the host ribosomes to gain free access for eIF2α that enables the prompt shutdown of host translation apparatus during virus infection. The studies reported here reveal a novel mechanism by which hantavirus NP prevents PKR induced host translation shutoff in virus infected cells. NP dissociates the inactive Hsp40-P58 IPK complex and recruits the released P58 IPK to the 40S ribosomal subunit, a strategic PKR location. This tactic recruitment of P58 IPK rapidly inhibits PKR and prevents host interference in viral protein synthesis.

Funding: MAM acknowledges the funding (1R15AI1128529-01) from NIH/NIAID to carry out the studies presented in this manuscript. SM and MAM also acknowledge the support in terms of research facilities and equipment provided by the Western University of Heath Sciences to carry out the studies reported in this manuscript. The funding organizations had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The P58 IPK , an endogenous PKR inhibitor [ 22 ] from the tetratricopeptide repeat (TPR) family, contains nine tandemly arranged repeats (TPR1-9) of 34 amino acids at the N-terminus. The homology between its C-terminal domain and the J domain of the DnaJ heat shock proteins also makes P58 IPK a member of the HSP family of proteins [ 23 ]. The P58 IPK inhibits PKR by direct binding and interruption in PKR dimerization [ 22 ]. The PKR binding domain of P58 IPK has been mapped to its TPR6 motif [ 22 ]. Interestingly, the activity of P58 IPK is itself negatively regulated by its own inhibitor Hsp40, which forms an inactive complex with P58 IPK (22). We have previously reported that hantavirus NP interrupts PKR dimerization by an unknown mechanism to ensure continuous synthesis of viral proteins in the host cell [ 24 ]. Here we demonstrate a novel mechanism by which hantavirus NP releases P58 IPK from the clutches of Hsp40 and recruits it to the 40S ribosomal subunit to inhibit PKR dimerization. Our results demonstrate that NP binds to P58 IPK with higher affinity as compared to Hsp40. Both NP and Hsp40 share a common binding site on P58 IPK . Our results suggest that NP competes with Hsp40 for binding to P58 IPK , leading to the dissociation of Hsp40-P58 IPK complex. The resulting NP-P58 IPK complex is selectively recruited to the 40S ribosomal subunit by the direct interaction between NP and the ribosomal protein S19 (RPS19), a structural component of the 40S ribosomal subunit. NP has distinct binding sites for RPS19 and P58 IPK . The simultaneous binding of NP to both the P58 IPK and RPS19 facilitates the prompt recruitment of P58 IPK to the 40S ribosomal subunit. Interruption in the recruitment process prevents PKR inhibition in cells. Collectively, our studies reveal a novel mechanism by which hantavirus NP releases P58 IPK from the clutches of its negative regulator and recruits it on the 40S ribosomal subunit to efficiently counteract the PKR antiviral responses.

Protein kinase R (PKR), one of the classical ISGs from the eIF2α-kinase family has a C-terminal kinase domain (KD) and an N-terminal dsRNA binding domain (dsRBD), composed of two tandem dsRNA binding motifs. The intermolecular interaction between dsRBD and KD maintains this enzyme in the latent form. Upon binding to dsRNA or its activator PACT protein, PKR undergoes a conformational change and forms a homo-dimer [ 12 , 13 ]. The dimerized PKR undergoes auto-phosphorylation to attain activity [ 14 ], which then plays diverse roles in the host antiviral defense. For example, the active PKR phosphorylates eIF2α to induce the host translation shutoff, aimed to interrupt the viral protein synthesis [ 15 ]. PKR also activates NF-κB to positively regulate the induction of interferon and inflammatory cytokines [ 16 ]. The activation of apoptotic pathways to limit virus replication and spread is yet another aspect of antiviral defense regulated by PKR [ 17 ]. The location of PKR inside the host cell might play a role in the diverse functions of PKR. Interestingly, PKR remains associated with the host cell ribosomes, especially the 40S ribosomal subunit [ 18 , 19 ]. The association with the ribosome has been proposed to facilitate the free access of PKR to its primary downstream target eIF2α, although the role of this strategic location in other antiviral functions of PKR remains unclear. Being a key antiviral host factor, viruses have evolved multiple strategies to antagonize PKR antiviral activity [ 20 , 21 ]. On the other hand, PKR activation is also strictly regulated by the host cell factors, such as, HIV-1 TAR RNA binding protein (TRBP), Glycoprotein p67, Nucleophosmin, Melanoma differentiation-associated gene-7 protein, Heat shock proteins Hsp90, Hsp70 and P58 IPK [ 14 ].

The virus-host interaction determines the outcome of a viral disease. The host type I interferon (IFN) response stimulates the expression of interferon stimulated genes (ISGs) [ 5 ]. The antiviral effector functions of ISGs promote the establishment of antiviral state in the host cell to create hurdles for virus replication [ 6 ]. Although the cytoplasmic tail domain of hantavirus Gn inhibits IFN induction during early stages of virus infection [ 7 – 9 ], a vigorous IFN and ISG expression is observed during later stages of hantavirus infection [ 10 ]. The delayed but robust IFN response fails to combat virus replication in hantavirus infected hosts [ 7 , 10 , 11 ], suggesting that hantaviruses have evolved strategies to counteract the ISGs’ antiviral effects.

Hantaviruses are zoonotic negative sense RNA viruses in the Hantaviridae family, Bunyavirales order. Their genome is composed of three RNA segments S, M and L, which encode nucleocapsid protein (NP), glycoprotein precursor (GPC) and RNA dependent RNA polymerase (RdRp), respectively [ 1 ]. The GPC is post-translationally cleaved into two glycoproteins Gn and Gc. Humans are infected by the inhalation of aerosolized excreta from virus-infected rodents [ 2 ]. Recently human to human transmission has been reported with the Andes hantavirus species in South America [ 3 ]. Hantavirus infections cause Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus cardiopulmonary syndrome (HCPS), having mortality rates of 15% and 40%, respectively [ 4 ]. Although 150,000 to 200,000 cases of hantavirus infection are annually reported worldwide, there is no antiviral therapeutic or FDA approved vaccine for this virus infection.

Results

NP binds to P58IPK and inhibits the PKR We previously reported that hantavirus NP inhibits PKR activation by inhibiting its dimerization and subsequent autophosphorylation. The inactive PKR fails to phosphorylate its downstream targets, such as, eIF2α. To determine whether NP uses P58IPK to inhibit the activation of PKR, we knocked down P58IPK by shRNA in HEK293T cells, stably expressing either NP from SNV (Sin Nombre virus) or EGFP as negative control. The cells were transfected with poly I:C three hours before harvesting to trigger PKR activation, and were examined by western blot analysis for the phosphorylation of PKR. It is evident from (Fig 1A and 1A1) that cells stably expressing NP failed to proficiently inhibit poly I:C induced PKR autophosphorylation due to the down regulation of P58IPK (compare lanes 6 and 8). The results were confirmed by using another shRNA targeted to a different region of P58IPK mRNA (Fig 1B and 1B1, compare lanes 4 and 6). This experiment suggests that NP requires P58IPK to proficiently inhibit the PKR. Unlike many other hantavirus NPs the NP from SNV does not block the RIGI/MDA5 mediated interferon β pathway [25,26], suggesting that potential inhibition of PKR activation through this route is less likely. To determine whether NP interacts with P58IPK, HEK293T cells were co-transfected with plasmids expressing FLAG tagged P58IPK and Myc tagged NP from either SNV or ANDV (Andes virus) or Hantaan virus or Myc tagged SUFU (suppressor of fused homolog) protein of chicken origin as negative control (Fig 1C lanes 2–5). The molecular weight of SUFU protein is similar to hantavirus N protein. Similarly, cells were co-transfected with plasmids expressing FLAG tagged P58IPK and NP from either SNV or ANDV or Hantaan virus devoid of any tag (Fig 1C, lanes 6–8). Cells were also co-transfected with plasmids expressing P58IPK devoid of any tag and Myc tagged NP from either SNV or ANDV or Hantaan virus or Myc tagged SUFU protein (Fig 1C, lanes 9–12). Cell lysates were immunoprecipitated using either anti-FLAG antibody or IgG as control. An examination of the immunoprecipitated material by western blot analysis using anti-Myc antibody revealed an interaction between P58IPK and NP from SNV, ANDV and Hantaan virus (Fig 1C). The reverse immunoprecipitation of cell lysates using anti-Myc antibody and examination of immunoprecipitated material by western blot analysis using anti-FLAG antibody further confirmed the results. The interaction of P58IPK with the NPs of all three hantaviruses (SNV, ANDV and Hantaan virus) is consistent with their previously reported PKR inhibition [24]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. NP recruits P58IPK to inhibit PKR activation. A and B. HEK293T stable cell lines expressing either EGFP or SNV NP were transduced with lentivirus for the stable expression of either scrambled shRNA or shRNA#1 (panel A) or shRNA#2 (panel B) against P58IPK. The shRNAs #1 and #2 were targeted to different regions of the P58IPK mRNA sequence. Cells were either mock transfected or transfected with Poly I:C (400 ng/ml) for three hours before harvesting. Cell lysates were examined for the expression of phosphorylated PKR (p-PKR), total PKR, P58IPK, NP, EGFP and β-actin by western blot analysis using appropriate antibodies. See Materials and Methods for antibodies used A1 and B1. The band intensities of p-PKR were quantified by imageJ and normalized related to the intensity in lane 2. The normalized intensity values were plotted verses lane number. The panels A1 and B1 represent the relative p-PKR band intensities from panels A and B, respectively. The standard deviations shown by error bars were calculated from two or more independent experiments. The significance of the difference was calculated by t test, as previously reported [24]. C. HEK293T cells were co-transfected with plasmids expressing the proteins shown in lanes 1–12. Cell lysates were immunoprecipitated (IP) with the antibodies shown in the figure, followed by western blot analysis (IB) of the immunoprecipitated material using the antibody shown in the figure. D. HUVEC cells were infected with either Andes virus or hazara virus. Cell lysates from infected cells were immunoprecipitated (IP) by the antibodies shown in the figure, except the IP of HZV NP was carried out using anti-CCHFV NP antibody as discussed in the text. The immunoprecipitated lysates were examined by western blot analysis (IB) using the antibodies shown in the figure. E. The bacterially expressed and purified HZV NP and ANDV NP were examined by western blot analysis using rabbit polyclonal anti-CCHFV antibody from abcam (ab190657). https://doi.org/10.1371/journal.ppat.1010007.g001 To further confirm the P58IPK-NP interaction, HUVECs were infected with either ANDV or Hazara virus (HZV), a tick borne orthonairovirus having tri-segmented negative sense RNA genome similar to hantaviruses. HZV is used as model virus for the study of Crimean congo hemorrhagic fever virus (CCHFV), a BSL 4 orthonairovirus that causes significant human disease. HUVECs were used because these cells are permissive to both hantavirus and HZV infection. The cell lysates were immunoprecipitated using either anti-ANDV NP antibody or IgG or anti-CCHFV NP antibody that cross reacts with HZV NP due to significant sequence homology between CCHFV and HZV NPs. We confirmed the cross reactivity of anti-CCHFV NP antibody with bacterially expressed and purified HZV NP (Fig 1E). These anti-CCHFV NP antibodies have been previously used for HZV NP studies [27]. The cross-reactive CCHFV antibodies were used because the antibodies for HZV NP are not commercially available. Western blot analysis of the immunoprecipitated material using anti-P58IPK antibody confirms the interaction between P58IPK and ANDV NP in infected cells (Fig 1D). The reverse immunoprecipitation of cell lysates using anti-P58IPK antibody, followed by Western blot analysis of the immunoprecipitated material using either anti-ANDV NP or anti-CCHFV NP antibodies further confirms the P58IPK-ANDV NP interaction during the course of infection (Fig 1D).

NP releases P58IPK from its negative regulator Hsp40 In the absence of viral infection or cell stress, the P58IPK forms an inactive complex with its negative regulator heat shock protein Hsp40. Since NP binds to P58IPK (Fig 1), we asked whether NP affects the binding of Hsp40 to P58IPK, which might lead to the dissociation of inactive P58IPK-Hsp40 complex. HUVEC cell lysates from ANDV or HZV infected cells were immunoprecipitated with anti-P58IPK antibody, followed by western blot analysis using anti-Hsp40 antibody. It is evident from Fig 1D that Hsp40-P58IPK interaction was significantly impacted in ANDV infected cells in comparison to HZV or un-infected control cells (compare lanes 7,8 and 9 in Fig 1D). To further confirm that NP impacted the Hsp40-P58IPK interaction, bacterially expressed and purified GST-P58IPK fusion protein was immobilized on Glutathione Sepharose beads. The beads were incubated with HEK293T cell lysates stably expressing either EGFP or NP, as mentioned in Materials and Methods. An examination of the GST pull-down material by western blot analysis revealed that Hsp40 strongly bound to GST-P58IPK in control cell lysates expressing EGFP. However, the interaction was impacted in cell lysates expressing NP (compare lane 6 with lane 3 in Fig 2A). The co-purification of both NP and Hsp40 with the GST-P58IPK suggests that NP likely competed with the Hsp40 for binding to GST-P58IPK. We next compared the binding of both NP and Hsp40 with the P58IPK in cells. HEK293T cells were co-transfected with plasmids expressing FLAG tagged P58IPK along with either Myc tagged NP or Myc tagged Hsp40. Cell lysates were immunoprecipitated with anti-Myc antibody and the binding of FLAG tagged P58IPK was examined by western blot analysis using anti-FLAG antibody. The strong co-purification of Myc-NP with FLAG-P58IPK suggests that NP likely binds to P58IPK more tightly as compared to Hsp40 (compare lanes 5 and 6 in Fig 2B). It is possible that both NP and Hsp40 share a common binding site on P58IPK and NP competes with Hsp40 for binding to P58IPK. Further studies will be required to demonstrate if NP competitively inhibits the binding of Hsp40 to P58IPK. It is equally possible that binding of NP induces a conformational change in P58IPK and conformationally altered P58IPK does not bind to Hsp40. PPT PowerPoint slide

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TIFF original image Download: Fig 2. NP releases P58IPK from its negative regulator Hsp40. A. Bacterially expressed and purified GST or GST-P58IPK fusion proteins were incubated with Glutathione Sepharose beads. The beads were washed and further incubated with the lysates from HEK293T cells, stably expressing either EGFP (lanes 1–3) or Myc-NP (lanes 4–6). The bound material eluted from washed beads was examined by western blot analysis using appropriate antibodies. Input represents the whole cell lysate. B. HEK293T cells were co-transfected with plasmids expressing FLAG-P58IPK along with either Myc-Hsp40 or Myc-NP. The cell lysates were immunoprecipitated using anti-Myc antibody. The western blot analysis of the whole cell lysate (WCL) (lanes 1–3) and immunoprecipiated material (lanes 4–6) was carried out using appropriate antibodies. C, D, E and F. Representative BLI sensograms showing over time association and dissociation of N protein with GST-P58IPK (C), N protein with GST tag (D), HSP40 with GST-P58IPK (E) and HSP40 with GST tag (F). The sensograms were generated at two concentrations of GST-P58IPK and GST tag, shown by red and blue color in panels C-F (see materials and methods for details). G. HEK293T cells were cotransfected with plasmids expressing FLAG-NP or Myc tagged wild type P58IPK or P58IPK mutants lacking either subdomain I composed of TPR repeats 1–3 (ΔTPR1-3) or both subdomains I and II composed of TPR repeats 1–6 (ΔTPR1-6). Cell lysates were Immunoprecipitated using anti-Myc antibody. Western blot analysis of the whole cell lysate (WCL) or immunoprecipitated material was carried out using either anti-FLAG or anti-Myc antibodies, as shown. H. The experiment shown in this panel was carried out exactly as in panel C except FLAG-NP was replaced with FLAG-Hsp40. I. Schematic representation of TPR domain of P58IPK. J. The kinetic profiles from panels C-F were analyzed for the calculation of binding parameters, as mentioned in Materials and Methods. Five percent of the calculated K D value was added as standard deviation. https://doi.org/10.1371/journal.ppat.1010007.g002 We next quantified the binding affinity of P58IPK with both NP and Hsp40 using biolayer interferometry on BLITZ (ForteBio) instrument, as previously reported [28,29]. Briefly, the bacterially expressed and purified C-terminally His tagged NP and Hsp40 were immobilized on Ni-NTA biosensor, following by the examination of association and dissociation kinetics of purified GST- P58IPK and GST (control) with the immobilized protein (see materials and methods for details). The representative kinetic profiles are shown in Fig 2C–2F and the binding data is shown in Fig 2J. It is evident from Fig 2C–2F that unlike GST control the GST-P58IPK showed interaction with both NP and Hsp40. The GST-P58IPK bound to NP with slower on-rate compared to Hsp40. However, the dissociation kinetics of GST- P58IPK -NP complex was slower compared to GST- P58IPK-Hasp40 complex. Analysis of the kinetic data reveled that GST- P58IPK bound to NP and Hsp40 with the dissociation constants (K D ) of ~ 0.8 μM and ~ 204 μM, respectively (Fig 2J). Similar binding affinities have been reported between most regulatory and signaling proteins [30–33]. This experiment clearly demonstrates that P58IPK binds to NP with 250-fold stronger affinity compared to Hsp40. We mapped the binding sites for NP and Hsp40 on P58IPK using deletion mutation and immunoprecipitation analysis. The N-terminal TPR domain of P58IPK is composed of nine TPR motifs (TPR1-TPR9). The structural motifs TPR1-TPR3, TPR4-TPR6, TPT7-TPR9 comprise three subdomains I, II and III, respectively (Fig 2I). Two P58IPK deletion mutants lacking either subdomain I (TPR1-TPR3) or both subdomains I and II (TPR1-TPR6) were expressed as N-terminally Myc tagged fusion proteins in HEK293T cells along with FLAG tagged NP. Cell lysates were immunoprecipitated with anti-Myc antibody and the binding of FLAG-NP was determined by western blot analysis using anti-FLAG antibody. It is evident from Fig 2G that deletion of subdomain I (ΔTPR1-3) significantly inhibited the binding of P58IPK to NP, which was further compromised by the deletion of both the subdomain I and II (ΔTPR1-6). This observation suggests that NP binding site is located in the subdomains I and II, although the subdomain I might play a major role in the binding. A similar experiment was performed to map the binding site of Hsp40 on P58IPK. It is evident from Fig 2H that deletion of subdomain I did not affect the binding of P58IPK to Hsp40. However, the binding was significantly compromised by the deletion of both the subdomains I and II, suggesting that Hsp40 binding site is most likely located in the subdomain II. Taken together, the results from Fig 2 demonstrate that NP binding site likely overlaps with the Hsp40 binding site on P58IPK. Since NP binds to P58IPK with higher affinity as compared to Hsp40 (Fig 2C–2F), it is possible that NP might competitively block the binding of Hsp40 to P58IPK, although further studies are required to prove a possible competitive inhibition. A weaker possibility that a host factor indirectly mediates the interaction between NP and P58IPK in vivo cannot be ruled out.

Oligomerization of NP is not required to inhibit PKR activation Hantavirus NP forms dimers and trimers upon expression, and the resulting monomeric, dimeric and trimeric forms of NP remain in dynamic equilibrium. Recently, the X-ray crystallographic studies have demonstrated that C-terminal linker and helix regions connecting the N-terminal coiled-coil domain and core region are essential for NP oligomerization [37]. Moreover, the electron microscopic (EM) visualization of native ribonucleoprotein complexes (RNPs) extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of the viral RNP [37], demonstrating that monomeric forms of NP are functional. We examined the oligomerization status of wildtype NP and its mutants on sucrose density gradient to determine whether oligomerization of NP is required to inhibit PKR activation. As shown in Fig 4F, the wildtype NP (~53 KDa) formed dimers (~106 KDa) and trimers (~159 KDa), and inhibited the PKR activation (Fig 4A, 4B and 4C). However, the NP mutant (NPΔ151–175) formed dimers and trimers similar to wildtype NP (Fig 4F) but failed to inhibit PKR activation (Fig 4D and 4D1). In comparison, the NP mutant (NP151-428) didn’t form the dimers and trimers (Fig 4F) due to the lack of N-terminal Coiled-coil domain, required for oligomerization, but inhibited the PKR activation (Fig 4C and 4C1). This clearly demonstrates that oligomerization of NP is not required to inhibit PKR activation.

NP recruits P58IPK to the 40S ribosomal subunit with the assistance of ribosomal protein RPS19 The observations from Figs 4 and 3D suggested that NP likely recruits P58IPK to the 40S ribosomal subunit, a previously known location of PKR. To test this hypothesis, HEK293T cells were co-transfected with plasmids expressing FLAG-P58IPK along with either Myc-NP or Myc-NPΔ151–175 deletion mutant. Cell lysates were immunoprecipitated with anti-Myc antibody. An examination of the immunoprecipitated material by western blot analysis revealed that wild type NP co-purified with both RPS19 and FLAG-P58IPK, demonstrating the possible recruitment of P58IPK to the 40S ribosomal subunit (Fig 5A). In comparison, the NPΔ151–175 mutant showed interaction with P58IPK but failed to bind RPS19, as expected. To further verify whether NP recruits P58IPK to the 40S ribosomal subunit, HEK293T cells were transfected as mentioned above. The 40S ribosomal subunit RPS19 was pulled down using anti-RPS19 antibody. An examination of the pulled-down material by western blot analysis revealed that RPS19 interacted with P58IPK in NP expressing cells (Fig 5B). This interaction was not observed in cells transfected with empty vector or a plasmid expressing NPΔ151–175 mutant (Fig 5B). These results suggest that NP recruits P58IPK to the 40S ribosomal subunit. To confirm that NP recruits P58IPK to the 40S ribosomal subunit in virus infected cells, HUVEC cell lysates from ANDV or HZV infected cells were immunoprecipitated with anti-P58IPK antibody. An examination of the immunoprecipitated material revealed that P58IPK in ANDV infected cells copurified with both NP and RPS19, demonstrating the recruitment of P58IPK to the 40S ribosomal subunit by the NP (Fig 1D). PPT PowerPoint slide

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TIFF original image Download: Fig 5. NP recruits P58IPK to the 40S ribosomal subunit. A. HEK293T cells were cotransfected with plasmids expressing FLAG-P58IPK along with either Myc-NP or Myc-NPΔ151–175 deletion mutant. Cell lysates were immunoprecipitated with anti-Myc antibody. The whole cell lysate (WCL) or immunoprecipitated material was examined by western blot analysis (IB) using either anti-RPS19 or anti-FLAG tag or anti-Myc tag antibodies. B. HEK293T cells were transfected as mentioned in panel A. The cell lysates were immunoprecipitated with RPS19 antibody. The whole cell lysate (WCL) or immunoprecipitated material was examined by western blot analysis (IB) using either anti-RPS19 or anti-FLAG tag or anti-Myc tag antibodies. C. HeLa cells were cotransfected with plasmids expressing mCherry-P58IPK along with either Myc-NP or Myc-NPΔ151–175 deletion mutant. The cells were examined by confocal microscope. The wild type NP and NPΔ151–175 mutant were visualized using anti-Myc primary antibody and a secondary antibody conjugated with Alexa Fluor 488. Quantification of the boxed zoomed area in the merged panels was carried out using imageJ as described in Fig 3D and presented as line graph at the bottom. The red and green fluorescence signals show strong colocalization evidenced by the significant increase in both fluorescence signals at the same location above the background. D. HeLa cells were cotransfected with mCherry-P58IPK expressing plasmid along with either empty vector or plasmids expressing wild type NP or NPΔ151–175 deletion mutant. The cells were examined by confocal microscope. The RPS19 was visualized using a primary anti-RPS19 antibody and a secondary antibody conjugated with Alexa Fluor 488. Quantification of the boxed zoomed area in the merged panels was carried out using imageJ as described in Fig 3D and presented sidewise as line graph. Unlike panels iii and v, the red and green fluorescence signals in panel iv show colocalization evidenced by the increase in both fluorescence signals at the same location above the background. https://doi.org/10.1371/journal.ppat.1010007.g005 We used confocal microscopy to further evaluate this hypothesis. It is evident from Fig 5C, that wild type NP shows a perinuclear punctate morphology, which is lost due to the deletion of RPS19 binding domain, as NPΔ151–175 deletion mutant is located all over the cytoplasm. This observation suggests that punctuate perinuclear morphology of wild type NP is likely due to its association with the 40S ribosomal subunit via RPS19. Although P58IPK strongly colocalized with both wildtype NP and NPΔ151–175 deletion mutant, the parental cytoplasmic morphology of P58IPK was altered into punctuate perinuclear morphology in NP expressing cells, resembling the morphology of wild type NP (Fig 5C), consistent with similar results from Fig 3D. This observation again suggests that NP likely recruits P58IPK to the 40S ribosomal subunit. To verify this observation, we examined the localization of mCherry-P58IPK fusion protein with RPS19, a structural constituent of the 40S ribosomal subunit, in cells expressing either wild type NP or NP deletion mutant. It is evident from Fig 5D that mCherry-P58IPK does not colocalize with RPS19 in cells lacking NP expression, suggesting that mCherry-P58IPK by itself does not bind to ribosomes. The colocalization between mCherry-P58IPK and RPS19 in NP expressing cells suggests that NP specifically mediates the interactions between P58IPK and the 40S ribosomal subunit. This is supported by the observation that NP deletion mutant deficient in binding to the 40S ribosome failed to mediate such interaction evident from the lack of strong colocalization between P58IPK and the RPS19 in cells expressing NPΔ151–175 deletion mutant (Fig 5D). To confirm that NP recruits P58IPK to the 40S ribosomal subunit with the assistance of RPS19, we asked whether P58IPK co-purifies with 40S ribosomal subunit in NP expressing cells. Cell lysates expressing either NP or NPΔ151–175 mutant or GFP, were fractionated on 5–40% sucrose gradient and ribosome sedimentation profiles were recorded based on the absorbance at 260 nm (A260) (Fig 6A). The 40S and 60S ribosomal subunits were monitored in the gradient fractions by the identification of RPS19 and RPL4, the structural components of the 40S and 60S ribosomal subunits, respectively (Fig 6B). The PKR was predominantly found in gradient fractions containing the 40S ribosomal subunit (Fig 6B). Interestingly, the P58IPK co-purified with the 40S ribosomal subunit along with NP and PKR (Fig 6B, middle panel). Such co-purification was not observed from cell lysates expressing either GFP or NPΔ151–175 mutant, which is deficient in binding to the RPS19, a structural component of the 40S ribosomal subunit (Fig 6B, top and bottom panels). This experiment clearly demonstrates that NP with the assistance of RPS19 recruits the P58IPK to the 40S ribosomal subunit where PKR is located. PPT PowerPoint slide

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TIFF original image Download: Fig 6. P58IPK cosedimented with 40S ribosomal subunit in the presence of NP. A. Ribosomal subunits sucrose density gradient fractionation. Absorbance of sucrose density gradient fractions at 260 nm was plotted verses fraction number. B. HEK293T cells were transfected with plasmids expressing EGFP, NP or NPΔ151–175 mutant. The cell lysates were fractionated on sucrose density gradient to generate the ribosomal subunit and polysomes sedimentation diagram, as shown in panel A. Panel B shows the western blot analysis of 40S and 60S ribosomal subunit associated proteins based on the plot diagram in panel A. Selected fractions were mixed with equal volume of 2x SDS sample buffer, boiled and subjected to SDS-PAGE. Input represents the cleared cell lysate. RPS19, RPL4, GFP, PKR, and P58IPK were probed with its specific antibodies. NP and NPΔ 151–175 were probed with anti-Myc tag antibody. https://doi.org/10.1371/journal.ppat.1010007.g006

[END]

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