One of the hallmarks of enterovirus infection is the inhibition of host cell protein synthesis through cleavage of eIF4G and PABP. A growing number of viruses are now known to cleave PABP during infection, including caliciviruses, hepatitis A virus and HIV, thus its evolutionary importance in virus replication is growing (Alvarez et al., 2006
; Kuyumcu-Martinez et al., 2004a
; Zhang et al, 2007
). While eIF4G is efficiently cleaved to completion early during PV infection, only a third of cytoplasmic PABP is cleaved at this time, and only 50–60% by late infection. Though partial PABP cleavage during viral infection may result from its huge abundance in cells, eluciation of the precise and restricted PABP-RNP complex(es) that is preferentially targeted by viral proteases is important to determine. We have shown that PABP is cleaved with biphasic kinetics by both viral proteinases, revealing a cleavage-resistant PABP population and we have shown that two PABP-binding proteins, eRF3 and Paip2, block PABP cleavage by two different proteinases.
Our results indicate that purified PABP inherently adopts conformations that are proteinase resistant. Because the PABP 3Cpro
cleavage sites do not perfectly match the 3Cpro
consensus cleavage site, incomplete PABP cleavage in cells could have resulted from reduced binding and catalytic rates. The results in show that 3Cpro
can easily cleave PABP with fast initial cleavage kinetics, indicating that 3Cpro
was amply active and that the non-consensus cleavage site sequence only minimally impedes cleavage. However, the abrupt reduction in PABP cleavage rate after 10–30 min suggests that at least two PABP pools exist in the population, one highly susceptible to cleavage and another configuration(s) that is refractory to cleavage. Slow secondary rate of cleavage by 3Cpro
suggests that interconversion between PABP conformations is inefficient. PABP cleavage by 2Apro
showed slower initial cleavage rates than 3Cpro
, but extended incubation time also revealed a biphasic cleavage profile. In these reactions, PABP likely interacts with itself through a well known, but poorly characterized oligomerization property (Kuhn and Pieler, 1996
). We have examined our PABP preparations by gel filtration and found anomalous migration (data not shown). Additionally, a recent report shows yeast PABP migrates in native gels as clear oligomers. This report also suggested that cysteine residues (conserved in human PABP) within the N-terminal RNA binding component of yeast PABP allow for a circular conformation to form in some PABP molecules through the formation of a disulfide bond. (Yao et al., 2007
). Such a configuration may block cleavage with 2Apro
, which cleaved more readily with high DTT concentration (), however 3Cpro
cleavage was not influenced by this parameter. This suggests that 2Apro
may recognize two different PABP pools. Importantly, biphasic PABP cleavage kinetics was also observed in HeLa extracts as well, however cleavage was consistently more efficient than with purified PABP. This suggests that factors present in lysates (e.g. polyA RNA) enhance PABP cleavage.
We previously reported that 3Cpro
primarily targets PABP molecules associated with the translational machinery, but molecular details were undetermined. Here we show that cleavage of PABP by viral proteinases is inhibited by Paip2 and eRF3, both of which function as translational repressors, but not by other the PABP-associated factors Tob1, Paip1, eIF4B, eIF4G, PCBP2 or unr ( and data not shown). eRF3 is proposed to transiently interact with polysome-bound PABP when ribosomes pause at stop codons, thus, eRF3 may only exert a minor inhibitory effect on overall PABP cleavage in a cell. However, other unknown interactions of eRF3 and PABP away from the context of translating polysomes remain possible. Paip2 is a general inhibitor of PABP function that can strip PABP off poly(A) RNA. This interaction is relatively stable and could interfere with PABP cleavage by steric effects and by reducing pools of poly(A)-bound PABP, which is more susceptible to 3Cpro
than free PABP (Khaleghpour et al., 2001b
; Kuyumcu-Martinez et al., 2002
). A new Paip2 homolog, termed Paip2B, has similar functions in regulating PABP and releasing it from poly(A) RNA (Berlanga et al., 2006
), thus it is likely that Paip2B can also inhibit the cleavage of PABP by viral proteinases.
Interestingly, Paip1 did not inhibit PABP cleavage, despite the fact it binds PABP at two sites that partly overlap the Paip2 binding sites. This difference could be attributed to the fact that the binding of Paip1 to PABP occurs with a 1:1 stoichiometry and with an apparent Kd of 1.9nM, whereas Paip2 binds PABP with a 2:1 stoichiometry and Kd values of 0.66 and 74nM (Khaleghpour et al. 2001
). Indeed, structural and thermodynamic studies of the binding of eRF3 and Paip1 or 2 peptides to the PABC domain of PABP indicated that binding of Paip1 causes a more drastic conformational change than Paip2 (Kozlov et al., 2004
; Kozlov et al., 2001
). Moreover, binding of Paip2 and eRF3 peptides (more than Paip1) is highly dependent on hydrophobic interactions or involves a larger protein/peptide contact surface (Kozlov et al., 2004
). Since proteinases probe subtrate structure, the differential cleavage results obtained with Paip1 and Paip2 suggest that PABP associated with these proteins adopts different conformations or that steric hindrance occurs only with Paip2.
Despite the finding that Paip2 can inhibit PABP cleavage by both proteinases and in all HeLa fractions tested, it probably plays a lesser role in modulating overall PABP cleavage in cells. Examination of actual protein concentrations in lysates indicated that Paip2 exists at levels approximately 50-fold lower than PABP, seemingly too low to account for the differential and incomplete PABP cleavage by 2Apro or 3Cpro in lysates and fractions thereof. 2Apro only cleaved PABP within slowly sedimenting complexes in polysome gradients, however, the bulk of the cellular Paip2 was already present in these same polysome gradient fractions, demonstrating insufficient endogenous Paip2 was present to block cleavage. Further, the PABP in these fractions could interact with Paip2 since addition of exogenous Paip2 strongly inhibited its cleavage with 2Apro ().
We also found an interesting inverse relationship between 2Apro and 3Cpro cleavage of PABP in HeLa cell fractions where PABP molecules within non-ribosome complexes (S200) were cleaved more efficiently by 2Apro than by 3Cpro. Conversely, PABP found within crude salt-washed ribosome extracts (SWRibo) was efficiently cleaved by 3Cpro, but not 2Apro (). This relationship partly extended to polysome fractions where 2A only cleaved non-ribosome associated PABP. The 2Apro cleavage site on PABP lies between the two 3Cpro sites in the linear sequence, yet no knowledge of true structure in this region is available, and the cleavage sites may actually lie in different surface regions. Our data show that each PV proteinase targets one of at least two distinct PABP populations, which present different conformational constraints. These unique conformations are likely modulated by differing host factors in complexes with PABP, which promote or inhibit cleavage of one or the other proteinase.
Why would 2Apro evolve to target non-ribosome-associated PABP but not 3Cpro? 3Cpro is the default picornavirus proteinase that has evolutionary homologs in other virus families such as norovirus, whereas 2Apro homologs are absent in many other virus families. Thus 3Cpro may have evolved early on to regulate viral and cellular translation and thus targets PABP in active polysomes. One possible function of the pool of PABP that is not associated with mRNA is to provide PABP for nascent RNA transcripts. Cleavage of this PABP population by 2Apro, the accessory proteinase, may serve to prevent nascent viral RNAs from acquiring intact PABP and being translated later in infection, thus aiding the process leading to packaging of vRNA. We have determined that PV virion RNA is completely devoid of PABP (data not shown).
PABP cleavage inhibits not only cap-dependent translation, but also PV translation (Bonderoff et al., 2008
) and HAV translation (Zhang et al., 2007
). Since viral translation persists for several hours longer than cellular translation in cells, we also investigated if viral proteinases preferentially target PABP cleavage on cellular versus viral polysomes as part of a mechanism that promotes viral translation. Surprisingly, we determined that PABP on either type of polysome pool was equally and highly susceptible to cleavage with 3Cpro
(). This suggests that viral polysomes do not stably bind factors that inhibit PABP cleavage. However, examination of PABP on active viral polysomes purified from infected cells shows that exclusively intact PABP was present; all PABP cleavage products migrated with 40–80S ribosome subunits ( and ). Thus, some form of PABP cleavage discrimination may exist in cells to restrict PABP cleavage on viral polysomes until after cleavage on cellular polysomes occurs. Also, viral proteinases must retain a population of intact PABP during the exponential phase of the viral growth cycle to support translation of the expanding pool of nascent viral RNAs. One hypothesis is that the non-poly(A) associated PABP which is resistant to 3Cpro
cleavage (S200 pool) may represent this population of intact PABP for nascent viral mRNA. Further, there was a temporal correlation between the downregulation of viral translation during late PV infection and appearance of PABP cps associating with viral polysomes (). This supports the hypothesis that intact PABP is required for viral translation.
We also investigated if 3Cpro or 3CD is the major proteinase that cleaves PABP. Since 3Cpro and 3CD had nearly equivalent cleavage activity on purified PABP, yet 3CD is more abundant in infected cells, 3CD may be dominant. However, RNA stimulated 3Cpro cleavage of PABP but not 3CD cleavage of PABP (), suggesting that 3Cpro is more effective versus poly(A)-bound PABP, which is presumably the more important target. These results make it unlikely that any preferential cleavage of cellular versus viral polysome-bound PABP could stem from regulation of 3CD processing into mature 3Cpro during the course of infection. However, since we have shown 3CD is an active PABP-specific proteinase, and was not inhibited by RNA, it likely plays a role in the cleavage of PABP on viral mRNA.
3CD may play an important role in the switch from viral translation to RNA replication, as it associates with the cloverleaf structure of the viral 5′ UTR and may mediate the circularization of the viral genome by interacting with PABP (Herold and Andino, 2001
). Since PABP cleavage, along with cleavage of PTB and PCBP2 (Back et al., 2002
; Perera et al., 2007
) all regulate the end of viral translation, 3CD and PCBP2 coordinately binding the viral cloverleaf and PABP may strongly stimulate PABP cleavage as well as cleavage of PCBP2 or PTB. We tested this hypothesis in vitro and did not observe a stimulation of cleavage activity when complexes of 3CD, PCBP2, PABP and viral minigenome RNA could form. It would be interesting to examine if addition of other viral replicase components 3AB, VPg-pUpU or 2C to this system can stimulate PABP or even PCBP2 cleavage.
In summary, this study provides insights about the mechanisms that modulate PABP cleavage during PV infection. PABP inherently exhibits a dual proteinase sensitive/resistant phenotype due to formation of multiple conformations, partly via self-oligomerization. Further, several types of PABP/protein/mRNP complexes influence these cleavage reactions but remain undefined at a molecular level. Interactions of PABP with eRF3 and Paip2 inhibit cleavage reactions with both viral proteinases, however, likely play a minor role in regulation of PABP cleavage during virus infection. More work remains to determine the primary PABP substrate complexes that 3CPro and 3CD have evolved to cleave most efficiently. Such complexes may exist only in a transient conformation that PABP adopts at a particular stage of the translation initiation, elongation or termination phases, or at a particular PABP moiety bound to poly(A).