A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression

A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.     

Translation of Sindbis virus 26S mRNA does not require intact eukariotic initiation factor 4G

The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.     

Translation Directed by Hepatitis A Virus IRES in the Absence of Active eIF4F Complex and eIF2

Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2A^pro or L^pro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2A^pro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) L^pro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2A^pro and L^pro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified L^pro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.     

Cleavage of eIF4G by HIV-1 protease: effects on translation

We have recently reported that HIV-1 protease (PR) cleaves the initiation factor of translation eIF4GI [Ventoso et al., Proc. Natl. Acad. Sci. USA 98 (2001) 12966-12971]. Here, we analyze the proteolytic activity of HIV-1 PR on eIF4GI and eIF4GII and its implications for the translation of mRNAs. HIV-1 PR efficiently cleaves eIF4GI, but not eIF4GII, in cell-free systems as well as in transfected mammalian cells. This specific proteolytic activity of the retroviral protease on eIF4GI was more selective than that observed with poliovirus 2A(pro). Despite the presence of an intact endogenous eIF4GII, cleavage of eIF4GI by HIV-1 PR was sufficient to impair drastically the translation of capped and uncapped mRNAs. In contrast, poliovirus IRES-driven translation was unaffected or even enhanced by HIV-1 PR after cleavage of eIF4GI. Further support for these in vitro results has been provided by the expression of HIV-1 PR in COS cells from a Gag-PR precursor. Our present findings suggest that eIF4GI intactness is necessary to maintain cap-dependent translation, not only in cell-free systems but also in mammalian cells.     

elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs.

Rhinovirus 2A and foot-and-mouth disease virus Lb proteinases stimulate the translation of uncapped messages and those carrying the rhinovirus and enterovirus Internal Ribosome Entry Segments (IRESes) by a mechanism involving the cleavage of host cell proteins. Here, we investigate this mechanism using an artificial dicistronic RNA containing the human rhinovirus IRES as intercistronic spacer. Because both proteinases cleave eukaryotic initiation factor 4G (eIF4G), we examined whether the cleavage products of eIF4G could stimulate uncapped or IRES-driven translation. Addition of intact eIF4F to translation extracts inhibited IRES-driven translation and reduced the translation stimulation observed in reactions pre-treated with Lb proteinase. Prolonged incubation of translation extracts with Lb proteinase removed all endogenous eIF4G and a substantial amount of the primary C- and N-terminal cleavage products. The translation of all mRNAs was reduced in such extracts. Capped mRNA translation was rescued by the addition of intact eIF4F. In contrast, addition of pre-cleaved eIF4F stimulated translation of uncapped or IRES-bearing messages to the levels seen upon proteinase addition. Furthermore, fractions containing the C-terminal, but not N-terminal, cleavage product of eIF4G stimulated translation moderately. These results demonstrate that the Lb and 2A proteinases stimulate translation of uncapped RNAs and those carrying IRESes by the production of cleavage products of eIF4G that enhance translation and by the removal of intact eIF4G that interferes with this stimulation.     

Eukaryotic initiation factor 4G-poly(A) binding protein interaction is required for poly(A) tail-mediated stimulation of picornavirus internal ribosome entry segment-driven translation but not for X-mediated stimulation of hepatitis C virus translation

Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5' m(7)GpppN cap and the 3' poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3' end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3'-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.     

Translation of viral mRNA without active eIF2: the case of picornaviruses

Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.     

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.     

Poliovirus 2A protease induces apoptotic cell death

A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.     

Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP

The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.     

Mutational analysis of the DEAD-box RNA helicase eIF4AII characterizes its interaction with transformation suppressor Pdcd4 and eIF4GI

Eukaryotic initiation factor (eIF) 4A unwinds secondary and tertiary structures in the 5'-untranslated region of mRNA, permitting translation initiation. Programmed cell death 4 (Pdcd4) is a novel transformation suppressor and eIF4A-binding partner that inhibits eIF4A helicase activity and translation. To elucidate the regions of eIF4A that are functionally significant in binding to Pdcd4, we generated point mutations of eIF4A. Two-hybrid analysis revealed that five eIF4A mutants completely lost binding to Pdcd4 while four eIF4A mutants retained wild-type levels of binding. The residues that, when mutated, inactivated Pdcd4 binding specified ATP binding, ATP hydrolysis, or RNA binding. With the exception of the Q-motif mutant eIF4AP56L, the eIF4A mutants inactivated for Pdcd4 binding were inactivated for binding to eIF4G (GM, GC, or both) and for enhancing translation. Several eIF4A mutants showing wild-type level binding to Pdcd4 were also inactivated for binding to eIF4G and for enhancing translation. Thus, significant dissociation of eIF4A's Pdcd4- and eIF4G-binding regions appears to occur. Because three of the four eIF4A mutants that retained Pdcd4 binding also suppressed translation activity in a dominant-negative manner, the structure that defines the Pdcd4-binding domain of eIF4A may be necessary but is insufficient for translation. A structural homology model of eIF4A shows regions important for binding to Pdcd4 and/or eIF4G lying on the perimeters of the hinge area of eIF4A. A competition experiment revealed that Pdcd4 competes with C-terminal eIF4G for binding to eIF4A. In summary, the Pdcd4-binding domains on eIF4A impact both binding to eIF4G and translation initiation in cells.     

The C-terminal domain of eukaryotic protein synthesis initiation factor (eIF) 4G is sufficient to support cap-independent translation in the absence of eIF4E.

The foot and mouth disease virus, a picornavirus, encodes two forms of a cysteine proteinase (leader or L protease) that bisects the EIF4G polypeptide of the initiation factor complex eIF4F into N-terminal (Nt) and C-terminal (Ct) domains. Previously we showed that, although in vitro cleavage of the translation initiation factor, eIF4G, with L protease decreases cap-dependent translation, the cleavage products themselves may directly promote cap-dependent protein synthesis. We now demonstrate that translation of uncapped mRNAs normally exhibits a strong requirement for eIF4F. However, this dependence is abolished when eIF4G is cleaved, with the Ct domain capable of supporting translation in the absence of the Nt domain. In contrast, the efficient translation of the second cistron of bicistronic mRNAs, directed by two distinct Internal Ribosome Entry Segments (IRES), exhibits no requirement for eIF4E but is dependent upon either intact eIF4G or the Ct domain. These results demonstrate that: (i) the apparent requirement for eIF4F for internal initiation on IRES-driven mRNAs can be fulfilled by the Ct proteolytic cleavage product; (ii) when eIF4G is cleaved, the Ct domain can also support cap-independent translation of cellular mRNAs not possessing an IRES element, in the absence of eIF4E; and (iii) when eIF4G is intact, translation of cellular mRNAs, whether capped or uncapped, is strictly dependent upon eIF4E. These data complement recent work in other laboratories defining the binding sites for other initiation factors on the eIF4G molecule.     

Cap-independent translation initiation in Xenopus oocytes.

Eukaryotic cellular mRNAs contain a cap at their 5'-ends, but some viral and cellular mRNAs bypass the cap-dependent mechanism of translation initiation in favor of internal entry of ribosomes at specific RNA sequences. Cap-dependent initiation requires intact initiation factor eIF4G (formerly eIF-4gamma, eIF-4Fgamma or p220), whereas internal initiation can proceed with eIF4G cleaved by picornaviral 2A or L proteases. Injection of recombinant coxsackievirus B4 protease 2A into Xenopus oocytes led to complete cleavage of endogenous eIF4G, but protein synthesis decreased by only 35%. Co-injection of edeine reduced synthesis by >90%, indicating that eIF4G-independent synthesis involved ongoing initiation. The spectrum of endogenous proteins synthesized was very similar in the presence or absence of intact eIF4G. Translation of exogenous rabbit globin mRNA, by contrast, was drastically inhibited by eIF4G cleavage. The N-terminal cleavage product of eIF4G (cpN), which binds eIF4E, was completely degraded within 6-12 h, while the C-terminal cleavage product (cpC), which binds to eIF3 and eIF4A, was more stable over the same period. Thus, translation initiation of most endogenous mRNAs inXenopusoocytes requires no eIF4G, or perhaps only cpC, suggesting a cap-independent mechanism.     

Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation

Translation of most eukaryotic mRNAs involves the synergistic action between the 5′ cap structure and the 3′ poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

The processing of eIF4GI by human rhinovirus type 2 2A(pro): relationship to self-cleavage and role of zinc

The 2A proteinase (2A(pro)) of human rhinoviruses (HRVs) is a cysteine protease containing a structurally important zinc ion. In the viral polyprotein, the enzyme cleaves between the C terminus of VP1 and its own N terminus. 2A(pro) also processes the two isoforms of the cellular protein, eukaryotic initiation factor 4G (eIF4G). We have shown that mature HRV2 2A(pro), when translated in vitro in rabbit reticulocyte lysates, efficiently cleaves eIF4GI, although the enzyme was not immediately active upon synthesis. Here, we examine the relationship between self-processing and eIF4GI cleavage. The onset of both reactions first occurred at least 10 min after initiation of protein synthesis. Furthermore, when self-processing was prevented by a specific mutation between VP1 and 2A(pro), the VP1-2A(pro) precursor was essentially unable to cleave eIF4GI, implying that self-processing is a prerequisite for eIF4GI cleavage. 2A(pro) synthesized in the presence of a potent zinc chelator is inactive; however, upon addition of excess zinc, HRV2 2A(pro) rapidly gained activity. Finally, the presence of the zinc chelator in the culture medium can protect HeLa cells from HRV infection.     

Interaction of translation initiation factor eIF4B with the poliovirus internal ribosome entry site

Poliovirus translation is initiated at the internal ribosome entry site (IRES). Most likely involving the action of standard initiation factors, this highly structured cis element in the 5" noncoding region of the viral RNA guides the ribosome to an internal silent AUG. The actual start codon for viral protein synthesis further downstream is then reached by ribosomal scanning. In this study we show that two of the secondary structure elements of the poliovirus IRES, domain V and, to a minor extent, domain VI, are the determinants for binding of the eukaryotic initiation factor eIF4B. Several mutations in domain V which are known to greatly affect poliovirus growth also seriously impair the binding of eIF4B. The interaction of eIF4B with the IRES is not dependent on the presence of the polypyrimidine tract-binding protein, which also binds to the poliovirus IRES. In contrast to its weak interaction with cellular mRNAs, eIF4B remains tightly associated with the poliovirus IRES during the formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. These results indicate that the interaction of eIF4B with the 3" region of the poliovirus IRES may be directly involved in translation initiation.     

Efficient cleavage of ribosome-associated poly(A)-binding protein by enterovirus 3C protease

Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2A(pro)) or 3C protease (3C(pro)). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3C(pro) is more efficient in cleaving PABP in ribosome-enriched fractions than 2A(pro) in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3C(pro)-mediated cleavage and inhibits 2A(pro)-mediated cleavage. These results suggest that 3C(pro) plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases.