Translational Enhancement of the Poliovirus 5' Noncoding Region
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Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 10272-10276, November 1992 Biochemistry Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A (tanslational regualon/dlistronlc mRNA/17 RNA ply e res cell me/firefly uferase gene) SIMON J. HAMBIDGE* AND PETER SARNOW*t Departments of *Microbiology and Immunology, and tBiochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, CO 80262 Communicated by Maxine F. Singer, July 6, 1992 ABSTRACT Genetic and biochemical studies have re- cleavage (7). As a result, eIF-4F is rendered nonfunctional, vealed that the 5' noncodlng region of pollovirus mites and capped host cell mRNAs cannot be translated by a tranation of the viral mRNA by an unusual m nim cap-dependent scanning mechanism (8), while the uncapped involving entry of ribosomes in internal sequences of mRNA viral mRNA can be translated by a cap-independent mech- molecules. We have found that mRNAs bearing the S' non- anism of internal ribosome binding (9). This mechanism coding region ofpoliovirus were transted at an enhced rate involves the binding ofribosomes to RNA sequences, termed in poliovirus-infected cells at a time when trans- the internal ribosome entry site (IRES) (10) or the ribosome lation of cellular mRNAs was not yet inhibited. This btnla- landing pad (9), present in the 5' NCR of the viral RNA. tional enhancement of the polioviral 5' noncodn region was Here, we provide evidence that mRNAs containing the 5' medated by the expression of virus-encoded polypeptde 2A. NCR of poliovirus are translated at an enhanced rate com- This indites that 2A Is a multifunctional protein Involved pared to mRNAs lacking the viral 5' NCR in cells at very directly or indirectly in the activation of viral mRNA trasla- early times after infection with poliovirus. Importantly, the tion, in addition to its known roles In viral pol i pro- observed enhanced rate of translation of poliovirus 5' NCR- cesing and in inhibition ofcellular protein synthesis. Thus, 2A containing RNAs occurred at a time when cellular translation represents an activator of tration of a viral mRNA that is was not inhibited, thus ruling out competition for the trans- translated by an inenal ribosoe bining mehi. A lation machinery as the only mechanism for the preferential likely consequence of this role of 2A is the effcient tra ion translation of viral RNA in infected cells. Furthermore, of viral mRNAs early in the infectious cycle, when host cell genetic analysis has identified the transactivator function as mRNAs can still compete with viral mRNAs for the host cell the poliovirus-encoded protein 2A. This finding indicates that translation apparatus. poliovirus encodes a translational transactivator function, possibly tojump-start viral gene expression very early in the Translational regulation in eukaryotic organisms can be me- infectious cycle, thereby circumventing competition with diated by sequences and structures in the RNA as well as ongoing cellular translation. proteins, causing both repression and activation of mRNA translation (for review, see ref. 1). Repression of translation initiation is usually mediated by RNA hairpin structures MATERIALS AND METHODS present in the 5' noncoding region (NCR) of mRNA mole- Cells and Viruses. Human HeLa cells and monkey COS cules. One well-studied example is the translational repres- cells (provided by R. Schneider, New York University) were sion offerritin mRNA, where the binding ofa 90-kDa protein grown on Petri dishes in Dulbecco's modified Eagle's me- to an RNA hairpin, the "iron responsive element," present dium supplemented with 10% (vol/vol) calf serum (GIBCO); in the 5' NCR of ferritin mRNA represses translation (2). OST7-1 cells (provided by 0. Elroy-Stein and B. Moss, Activation of translation has been reported in Saccharomy- Laboratory of Viral Diseases, National Institutes of Health, ces cerevisiae. For example, the regulation of translation of Bethesda, MD) were grown in modified Eagle's medium GCN4 mRNA by the GCN and GCD gene products is supplemented with 10% (vol/vol) fetal bovine serum and dependent on the availability ofamino acids (3). In this case, G418 sulfate (Geneticin; GIBCO) at 0.5 mg/ml. Preparation some of the translational regulators (for example, the eu- of poliovirus type 1 stocks and protocols for viral infections karyotic translation factor 2) have been shown to be com- were as described (11). ponents of the normal translation apparatus (4). Recently, Plasmds. Plasmid pSV-TK/P2/CAT was provided by N. translational transactivation of viral mRNAs by virus- Sonenberg (McGill University, Montreal). The constructions encoded gene products has been reported. The translation of of plasmids T7-Sncpolio-LUC and T7-Sncluc-LUC (12), T7- the 35S mRNA of cauliflower mosaic virus was found to be POLIO (13), T7-3NC-202 (14), and T7-2B-201 (11) have been stimulated by the gene product of the viral 19S mRNA (5). described. Plasmid T7-OILOP was obtained from K. Kirke- This finding indicates that viruses can carry functions to gaard (University of Colorado, Boulder). Plasmid T7-TMEV enhance the translation of their genetic material, thus cir- (strain BeAn) was provided by A. Pritchard and H. Lipton cumventing competition for the translation apparatus with (Department of Neurology, University of Colorado Health host cell mRNAs. Science Center, Denver). Plasmid T7-2A-1 was constructed Poliovirus, a positive-strand RNA virus, has been shown to by ligation ofthree fragments: an Ava I/Bgl II fragment (base inhibit host cell translation by cleavage ofthe p220 protein of pairs 2978-5601) from pSV2-polio (2A-1) (15), aBgl II/EcoRI the cap-binding complex eIF-4F (6). Subsequently, it was fragment (base pairs 5601-7498) from pSV2-polio (15), and a found that the virus-encoded protein 2A, together with the 5.3-kilobase EcoRIf/Ava I fragment from T7-POLIO (13). translation initiation factor eIF3, is responsible for p220 T7-P1/2A was constructed by deleting base pairs 3915-7000 The publication costs ofthis article were defrayed in part by page charge Abbreviations: NCR, noncoding region; IRES, internal ribosome payment. This article must therefore be hereby marked "advertisement" entry site(s); CAT, chloramphenicol acetyltransferase; TK, thymi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dine kinase; BiP, immunoglobulin heavy-chain binding protein. 10272 Downloaded by guest on September 30, 2021 Biochemistry: Hambidge and Sarnow Proc. Nati. Acad. Sci. USA 89 (1992) 10273 (a HincIl fragment) from T7-POLIO. Subsequently, T7- chloramphenicol acetyltransferase (CAT) as a second cis- 5ncpolio-2A was made by deleting base pairs 865-3082 (a tron. The first cistron, TK, should therefore be translated by BstBI fragment) from T7-P1/2A. The vectors pSV-CAT/ a cap-dependent scanning mechanism, whereas the second 5ncpolio/LUC and pSV-CAT/5ncbip/LUC have been de- cistron should be translated by an internal ribosome binding scribed (16). pCMV-5ncbip-LUC contains an immediate mechanism mediated by the polioviral 5' NCR (9). early promoter/enhancer element from human cytomegalo- The pSV-TK/P2/CAT construct was expressed as pre- virus upstream ofthe 5' NCR ofimmunoglobulin heavy-chain dicted (9), and transfected cells were either mock-infected or binding protein (BiP) and the luciferase (LUC) coding region infected with poliovirus and labeled with [35S]methionine. (a gift from D. G. Macejak, University of Colorado Health Soluble extracts were prepared and analyzed by SDS/PAGE. Science Center, Denver). Fig. 1 shows that the lysate from mock-infected cells (lane 1) Preparation ofRadiolabeled Extracts from Cells Transfected displayed the same rate of translation of cellular mRNAs as with Plasmids. Five micrograms of supercoiled plasmid DNA the lysate obtained from infected cells (lane 2). Thus, this was transfected into 106 COS cells by using a calcium experimental protocol permitted us to test the translational phosphate precipitation method as described (17). Forty efficiency of mRNAs under conditions in which overall host hours after transfection, the cells were mock-infected or cell translation was not yet inhibited due to poliovirus infec- infected with poliovirus at a multiplicity of infection of 50. tion. Next the lysates were immunoprecipitated with an After an incubation for an additional 2.125 hr, the cells were antibody directed against TK protein. Again, similar amounts radiolabeled by replacing the medium with methionine-free of TK protein were present in lysates from mock-infected medium containing [35S]methionine [400 ,ACi/ml (1 Ci = 37 that GBq); New England Nuclear] and incubated for an additional (lane 3) and poliovirus-infected cells (lane 4), indicating hour. Cells were then washed three times with ice-cold the cap-dependent translation of the first cistron in the phosphate-buffered saline and lysed into 500 M1 of ice-cold dicistronic reporter mRNA was not affected under these lysis buffer (50 mM Tris-HCI, pH 7.9/5 mM EDTA/150 mM conditions. NaCI/1% Nonidet P-40). Extracts were subjected to centrif- In poliovirus-infected cells, however, translation of the ugation (12,000 x g for 5 min), and the soluble supernatants second cistron, CAT, mediated by internal ribosome binding were used as the source for labeled proteins. to the 5' NCR of poliovirus, was markedly enhanced in Cotransfections of Plasmids into Mouse OST7-1 Cells. infected extracts compared to mock-infected extracts (lanes Dishes containing 106 OST7-1 cells were transfected as 6 and 5, respectively). Analysis by scanning densitometry described above, except that 1 ,ug of reporter DNA (see revealed that 8-fold more CAT protein was labeled in infected Results) was used either alone or with 10 ,ug of effector DNA extracts than in mock-infected extracts. Since the enhanced (see Results). The cells were shocked with 10% (vol/vol) translation of the second cistron mediated by the 5' NCR of glycerol (in Dulbecco's modified Eagle's medium) for 90 sec 1 2 3 4 5 at 4 hr after transfection and harvested 2 hr later by scraping the cells with a rubber policeman into 1 ml of phosphate- buffered saline without calcium and magnesium.