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Regulación de la traducción mediada por la proteína NSP3 de rotavirus

The study of the mechanisms used by different viruses to control host cell translation and favor the expression of viral proteins has been very instructive to know fundamental cellular mechanisms that regulate translation. Species A rotavirus (RVA) NSP3 protein is capable of inhibiting the translation of cellular mRNAs as a consequence of its binding to the translation initiation factor eIF4G and of stimulating the translation of viral mRNAs as a consequence of its binding to the UGACC sequence at their 3’-end. It has been proposed that NSP3 inhibits translation by interference with the circularization of the 5’-3’-ends of cellular mRNAs mediated by eIF4E-eIF4G-PABP (poly-A binding protein), and simultaneously it has been proposed that NSP3 stimulates translation of RVA mRNAs by circularization, in a way analogous to that performed by PABP on cellular mRNAs. However, the importance of NSP3 dimerization assisted by chaperone HSP90 in its inhibitory function of cell translation is unknown. Recently, the importance of NSP3 dimerization intermediates on its inhibitory function on host cell translation was explored, and it was found that point mutations in the coiled-coil region affect the formation of NSP3 dimers and partially preserve its host cell translation inhibitory function. In addition, it was found that NSP3 dimers degrade more rapidly than dimerization intermediates. These data demonstrate that the function of NSP3 is acquired prior to the appearance of dimers and suggests that the proteasome susceptibility of the different oligomeric forms of NSP3 are relevant in the establishment of the inhibitory function of cell translation.El estudio de los mecanismos utilizados por diversos virus para controlar la traducción de la célula hospedera y favorecer la expresión de proteínas virales ha sido muy instructivo para conocer mecanismos celulares fundamentales que regulan la traducción. La proteína NSP3 de los rotavirus de la especie A (RVA) es capaz de inhibir la traducción de los RNAm celulares como consecuencia de su unión al factor de iniciación de la traducción eIF4G y de estimular la traducción de los RNAm virales como consecuencia de su unión a la secuencia UGACC de su extremo 3’. Se ha propuesto que NSP3 inhibe la traducción por interferir con la circularización de los extremos 5’-3’ de los RNAm celulares mediada por eIF4E-eIF4G-PABP (proteína de unión a poli-A), y simultáneamente se propone que NSP3 estimula la traducción de los RNAm del RVA circularizándolos de manera análoga a la que realiza PABP en los RNAm celulares. Sin embargo, la importancia de la dimerización de NSP3 asistida por la chaperona HSP90 en su función inhibitoria de la traducción celular se desconoce. Recientemente, se exploró la importancia de los intermediarios de la dimerización de NSP3 sobre su función inhibitoria de la traducción, y se encontró que mutaciones puntuales en la región coiled-coil afectan la formación de los dímeros y conservan parcialmente su función inhibitoria de la traducción celular. Además, se detectó que los dímeros de NSP3 se degradan con mayor rapidez que los intermediarios de la dimerización. Estos datos demuestran que la función de NSP3 se adquiere previamente a la aparición de los dímeros, y sugiere que la susceptibilidad al proteasoma de las distintas formas oligoméricas de NSP3 son relevantes en el establecimiento de la función inhibitoria de la traducción celular.

Species A rotavirus NSP3 acquires its translation inhibitory function prior to stable dimer formation

<div><p>Species A rotavirus non-structural protein 3 (NSP3) is a translational regulator that inhibits or, under some conditions, enhances host cell translation. NSP3 binds to the translation initiation factor eIF4G1 and evicts poly-(A) binding protein (PABP) from eIF4G1, thus inhibiting translation of polyadenylated mRNAs, presumably by disrupting the effect of PABP bound to their 3’-ends. NSP3 has a long coiled-coil region involved in dimerization that includes a chaperone Hsp90-binding domain (HS90BD). We aimed to study the role in NSP3 dimerization of a segment of the coiled-coil region adjoining the HS90BD. We used a vaccinia virus system to express NSP3 with point mutations in conserved amino acids in the coiled-coil region and determined the effects of these mutations on translation by metabolic labeling of proteins as well as on accumulation of stable NSP3 dimers by non-dissociating Western blot, a method that separates stable NSP3 dimers from the monomer/dimerization intermediate forms of the protein. Four of five mutations reduced the total yield of NSP3 and the formation of stable dimers (W170A, K171E, R173E and R187E:K191E), whereas one mutation had the opposite effects (Y192A). Treatment with the proteasome inhibitor MG132 revealed that stable NSP3 dimers and monomers/dimerization intermediates are susceptible to proteasome degradation. Surprisingly, mutants severely impaired in the formation of stable dimers were still able to inhibit host cell translation, suggesting that NSP3 dimerization intermediates are functional. Our results demonstrate that rotavirus NSP3 acquires its function prior to stable dimer formation and remain as a proteasome target throughout dimerization.</p></div

A model of RVA NSP3 translation inhibition prior to stable dimer formation.

<p>The current model (above) propose that mature NSP3 dimers engage eIF4F-mRNA-PABP complexes thus disrupting 5’ to 3’ mRNA interaction. Alternatively, NSP3 dimerization intermediates bound to HSP90 (below) would sequester eIF4G1.</p

Mutations produced in the coiled-coil region of the RRV NSP3 gene and comparison with the sequence of other RVA strains.

<p>(A) Schematic representation of NSP3 domains: RBD, RNA-binding; RoBD, RoXaN-binding; HID, two-hybrid interaction; HS90BD, Hsp90-binding; GBD, eIF4G1-binding. The lower line indicates the coiled-coil region, and the numbers refer to the NSP3 amino acid sequence. (B) Alignment of the amino acid sequences of the coiled-coil region of the NSP3 gene of RRV and other RVA strains that infect diverse animal species. The heptad repeat pattern (a-g)_n is shown above, where “a” and “d” are hydrophobic and “e” and “g” are charged residues. The remaining positions, “b”, “c” and “f”, tend to be occupied by polar residues. Mutagenized residues are highlighted in red. The lower lines indicate the segments of the coiled-coil region that overlap with NSP3 domains.</p

Determination of the half-lives of stable dimeric and monomeric/dimerization intermediate forms of wtNSP3 in the presence and absence of MG132.

<p>Identical numbers of BSC1 cells were infected with vNSP3 to express wtNSP3. At two hpi, the cells were treated with MG132 or its diluent DMSO, and at 14 hpi cycloheximide (CHX) was added to inhibit translation. The cells were harvested 0, 30, 60, 120 and 240 min after adding CHX. The harvested cells were analyzed by WB-ND with anti-NSP3 serum (A). The two bands correspond to the monomeric/dimerization intermediate (34 kDa) and dimeric (68 kDa) forms of NSP3. Based on densitometry analysis of three independent experiments, the graph lines (B) were used to determine the half-lives of the monomeric/dimerization intermediate (134 min) and stable dimeric (60 min) forms of the protein in the absence of MG132. Both forms of the protein were more stable in the presence of MG132. Bar lines indicate standard deviation.</p

Effect of point mutations in the coiled-coil region of NSP3 on the accumulation of its stable dimeric and monomeric/dimerization intermediate forms in the presence and absence of MG132.

<p>Identical numbers of BSC-1 cells were infected with vaccinia viruses for the expression of wild type NSP3 or its mutants. The cells were incubated with a proteasome inhibitor (MG132) or its diluent (DMSO). The infected cells were harvested at 18 hpi and analyzed by WB-ND with anti-NSP3 serum (A and B). The two prominent bands correspond to monomeric/dimerization intermediate (34 kDa) and dimeric (68 kDa) forms of NSP3; the minor band may correspond to a putative degradation product (30 kDa). Based on densitometry analysis of three independent experiments, the graph bars compare the accumulation of monomers/dimerization intermediates (C) or stable NSP3 dimers (D) in the presence of MG132 (white bars) or its diluent (gray bars). Bars indicate standard deviation.</p

Effect of point mutations in the coiled-coil region of RRV NSP3 on its accumulation and susceptibility to the proteasome.

<p>Identical numbers of BSC-1 cells were infected with the parental virus vT7lacOI or with viruses for the expression of wild type NSP3 or its mutants. The cells were incubated with a proteasome inhibitor (MG132) or with its diluent (DMSO). The infected cells were harvested at 18 hpi and analyzed by WB with anti-NSP3 serum (A). Based on densitometry analysis of three independent experiments, the graph bars (B) indicate the accumulation of NSP3 in the presence of MG132 (white bars) or its diluent DMSO (gray bars) compared with the accumulation of the wild type protein in the absence of MG132 (Wt). Bars indicate standard deviation.</p

Effect of point mutations in the coiled-coil region of RRV NSP3 on the inhibitory function of NSP3 in host cell translation.

<p>BSC-1 cells were mock-infected, infected with the parental virus vT7lacOI, or with viruses for the expression of wild type NSP3 or its mutants with a MOI of five. At two hpi the inducer IPTG was added (0.4 mM). The infected cells were pulse labeled with [³⁵S]-methionine plus [³⁵S]-cysteine prior to harvesting at 18 hpi. The cells were then analyzed by SDS-PAGE and autoradiography (A). The molecular weights of four predominant vaccinia virus proteins detected in lane 2 are indicated to the left. The solid triangle indicates the position of the NSP3 bands (34 kDa). Based on densitometry analysis of three independent experiments, the graph bars (B) indicate the percentage of protein synthesis in cells expressing NSP3 or its mutants compared with control cells that do not express NSP3 (vT7). Bars indicate standard deviation.</p