poly(A)-binding Protein Cleavage Research Articles

Cleavage of poly(A)-binding protein by duck hepatitis A virus 3C protease

During viral infections, some viruses subvert the host proteins to promote the translation or RNA replication with their protease-mediated cleavage. Poly (A)-binding protein (PABP) is a target for several RNA viruses; however, the impact of duck hepatitis A virus (DHAV) on PABP remains unknown. In this study, we demonstrated for the first time that DHAV infection stimulates a decrease in endogenous PABP and generates two cleavage fragments. On the basis of in vitro cleavage assays, an accumulation of PABP cleavage fragments was detected in duck embryo fibroblast (DEF) cell extracts incubated with functional DHAV 3C protease. In addition, DHAV 3C protease was sufficient for the cleavage of recombinant PABP without the assistance of other eukaryotic cellular cofactors. Furthermore, using site-directed mutagenesis, our data demonstrated a 3C protease cleavage site located between Q367 and G368 in duck PABP. Moreover, the knockdown of PABP inhibited the production of viral RNA, and the C-terminal domain of PABP caused a reduction in viral replication compared to the N-terminal domain. Taken together, these findings suggested that DHAV 3C protease mediates the cleavage of PABP, which may be a strategy to manipulate viral replication.

Site-Specific Cleavage of the Host Poly(A) Binding Protein by the Encephalomyocarditis Virus 3C Proteinase Stimulates Viral Replication

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3C(pro) cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3C(pro) cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication.

HIV- 1 Protease Inhibits Cap- and Poly(A)-Dependent Translation upon eIF4GI and PABP Cleavage

A number of viral proteases are able to cleave translation initiation factors leading to the inhibition of cellular translation. This is the case of human immunodeficiency virus type 1 protease (HIV-1 PR), which hydrolyzes eIF4GI and poly(A)-binding protein (PABP). Here, the effect of HIV-1 PR on cellular and viral protein synthesis has been examined using cell-free systems. HIV-1 PR strongly hampers translation of pre-existing capped luc mRNAs, particularly when these mRNAs contain a poly(A) tail. In fact, HIV-1 PR efficiently blocks cap- and poly(A)-dependent translation initiation in HeLa extracts. Addition of exogenous PABP to HIV-1 PR treated extracts partially restores the translation of polyadenylated luc mRNAs, suggesting that PABP cleavage is directly involved in the inhibition of poly(A)-dependent translation. In contrast to these data, PABP cleavage induced by HIV-1 PR has little impact on the translation of polyadenylated encephalomyocarditis virus internal ribosome entry site (IRES)-containing mRNAs. In this case, the loss of poly(A)-dependent translation is compensated by the IRES transactivation provided by eIF4G cleavage. Finally, translation of capped and polyadenylated HIV-1 genomic mRNA takes place in HeLa extracts when eIF4GI and PABP have been cleaved by HIV-1 PR. Together these results suggest that proteolytic cleavage of eIF4GI and PABP by HIV-1 PR blocks cap- and poly(A)-dependent initiation of translation, leading to the inhibition of cellular protein synthesis. However, HIV-1 genomic mRNA can be translated under these conditions, giving rise to the production of Gag polyprotein.

Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

The two enteroviral proteinases, 2A proteinase (2A(pro)) and 3C proteinase (3C(pro)), induce host cell translation shutoff in enterovirus-infected cells by cleaving canonical translation initiation factors. Cleavage of poly(A)-binding protein (PABP) by 3C(pro) has been shown to be a necessary component for host translation shutoff. Here we show that 3C(pro) inhibits cap-independent translation mediated by the poliovirus internal ribosome entry site (IRES) in a dose-dependent manner in HeLa translation extracts displaying cap-poly(A) synergy. This effect is independent of the stimulatory effect of 2A(pro) on IRES translation, and 3C(pro)-induced translation inhibition can be partially rescued by addition of recombinant PABP in vitro. 3C(pro) inhibits IRES translation on transcripts containing or lacking poly(A) tails, suggesting that cleavage of PABP and IRES trans-activating factors polypyrimidine tract-binding protein and poly r(C)-binding protein 2 may also be important for inhibition. Expression of 3C(pro) cleavage-resistant PABP in cells increased translation of nonreplicating viral minigenome reporter RNAs during infection and also delayed and reduced virus protein synthesis from replicating RNA. Further, expression of cleavage-resistant PABP in cells reduced the accumulation of viral RNA and the output of infectious virus. These results suggest that cleavage of PABP contributes to viral translation shutoff that is required for the switch from translation to RNA replication.

Modulation of enteroviral proteinase cleavage of poly(A)-binding protein (PABP) by conformation and PABP-associated factors

Poliovirus (PV) causes a drastic inhibition of cellular cap-dependant protein synthesis due to the cleavage of translation factors eukaryotic initiation factor 4G (eIF4G) and poly(A) binding protein (PABP). Only about half of cellular PABP is cleaved by viral 2A and 3C proteinases during infection. We have investigated PABP cleavage determinants that regulate this partial cleavage. PABP cleavage kinetics analyses indicate that PABP exists in multiple conformations, some of which are resistant to 3C pro or 2A pro cleavage and can be modulated by reducing potential. Cleavage reactions containing a panel of PABP-binding proteins revealed that eukaryotic release factor 3 (eRF3) and PABP-interacting protein 2 (Paip2) modulate and interfere with the cleavage susceptibility of PABP, whereas all other PABP-binding proteins tested do not. We show that PABP on cellular polysomes is cleaved only by 3C pro and that Paip2 does not sediment with polysomes. Also, viral polysomes contained only full-length PABP, however, cellular or viral ribosomes were equally susceptible to 3C pro cleavage in vitro. Finally, we determined that precursor 3CD and mature 3C pro have equivalent cleavage activity on purified PABP, but only 3C pro cleavage activity was stimulated by PABP-binding viral RNA. The results further elucidate complex mechanisms where multiple inherent PABP conformations and protein and RNA interactions both serve to differentially regulate PABP cleavage by 3CD, 3C pro and 2A pro.

Poly(A) binding protein, C-terminally truncated by the hepatitis A virus proteinase 3C, inhibits viral translation

Proteolytic cleavage of translation initiation factors is a means to interfere with mRNA circularization and to induce translation arrest during picornaviral replication or apoptosis. It was shown that the regulated cleavages of eukaryotic initiation factor (eIF) 4G and poly(A)-binding protein (PABP) by viral proteinases correlated with early and late arrest of host cap-dependent and viral internal ribosome entry site (IRES)-dependent translation, respectively. Here we show that in contrast to coxsackievirus, eIF4G is not a substrate of proteinase 3C of hepatitis A virus (HAV 3Cpro). However, PABP is cleaved by HAV 3Cpro in vitro and in vivo, separating the N-terminal RNA-binding domain (NTD) of PABP from the C-terminal protein-interaction domain. In vitro, NTD has a dominant negative effect on HAV IRES-dependent translation and an enhanced binding affinity to the RNA structural element pY1 in the 5′ nontranslated region of the HAV RNA that is essential for viral genome replication. The results point to a regulatory role of PABP cleavage in RNA template switching of viral translation to RNA synthesis.

Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

Cleavage of poly(A)-binding protein by poliovirus 3C protease inhibits host cell translation: a novel mechanism for host translation shutoff.

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) by viral 2A protease (2Apro) has been proposed to cause severe translation inhibition in poliovirus-infected cells. However, infections containing 1 mM guanidine-HCl result in eIF4GI cleavage but only partial translation shutoff, indicating eIF4GI cleavage is insufficient for drastic translation inhibition. Viral 3C protease (3Cpro) cleaves poly(A)-binding protein (PABP) and removes the C-terminal domain (CTD) that interacts with several translation factors. In HeLa cell translation extracts that exhibit cap-poly(A) synergy, partial cleavage of PABP by 3Cpro inhibited translation of endogenous mRNAs and reporter RNA as effectively as complete cleavage of eIF4GI and eIF4GII by 2Apro. 3Cpro-mediated translation inhibition was poly(A) dependent, and addition of PABP to extracts restored translation. Expression of 3Cpro in HeLa cells resulted in partial PABP cleavage and similar inhibition of translation. PABP cleavage did not affect eIF4GI-PABP interactions, and the results of kinetics experiments suggest that 3Cpro might inhibit late steps in translation or ribosome recycling. The data illustrate the importance of the CTD of PABP in poly(A)-dependent translation in mammalian cells. We propose that enteroviruses use a dual strategy for host translation shutoff, requiring cleavage of PABP by 3Cpro and of eIF4G by 2Apro.

Degradation of poly(A)-binding protein in apoptotic cells and linkage to translation regulation.

We have recently shown that poly(A)-binding protein (PABP) is cleaved during poliovirus and Coxsackievirus infection by viral 3Cprotease and that 3Cprotease modification of a subset of PABP can result in significant translation inhibition. During apoptosis, translation undergoes significant down-regulation that correlates with caspase-3 mediated cleavage of several translation factors, including eIF4G, 4EBP1 and eIF2alpha. The fate of PABP in apoptotic cells has not yet been examined. Here we show that PABP levels decline significantly via proteolytic degradation in apoptotic HeLa, Jurkat and MCF7 cells. The degradation of PABP correlated with translation inhibition but lagged behind cleavage of eIF4GI. In apoptotic MCF7 cells translation inhibition occurred without modification of most translation factors and correlated with PABP degradation. PABP was not cleaved during incubation with several caspases, yet caspase 3 induced weak PABP degradative activity in cells lysates. Both the caspase inhibitor zVAD and calpain inhibitors blocked PABP cleavage in vivo, while the proteosome inhibitor MG132 induced PABP degradation. Protease(s) activated during apoptosis preferentially degraded PABP associated with ribosomes and translation factors, but not PABP in other cellular compartments. The data suggest that targeted degradation of PABP contributes to translation inhibition in apoptotic cells.

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.

Cleavage of poly(A)-binding protein by enterovirus proteases concurrent with inhibition of translation in vitro.

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3' end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5'-end-3'-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.

Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro and in vivo: another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.