The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender.

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 106 and 800 × 106 spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

Int6 regulates both proteasomal degradation and translation initiation and is critical for proper formation of acini by human mammary epithelium.

INT6/EIF3E has been implicated in breast tumorigenesis, but its functional activities remain poorly defined. We found that, repressing INT6 expression induced transformed properties in normal human mammary epithelium (MCF10A); in contrast, Int6 silencing induced apoptosis in HeLa cells. As in fission yeast, Int6 in human cells was required for assembly of active proteasomes. A reverse-phase protein array screen identified SRC3/AIB1 as one oncoprotein the level and stability of which increased when Int6 was silenced in MCF10A cells. Our data further show that Int6 binds SRC3 and its ubiquitin ligase Fbw7, thus perhaps mediating the interaction between SRC3-Fbw7 and proteasomes. Consistent with this, Int6 silencing did not increase SRC3 levels in HeLa cells, which have low Fbw7 levels. It is surprising that, however, polyubiquitylated proteins do not accumulate or may even decrease in Int6-silenced cells that contain defective proteasomes. Considering that decreased ubiquitin might explain this observation and that Int6 might control ubiquitin levels in its role as a subunit of eIF3 (eukaryote translation initiation factor 3), we found that silencing Int6 reduced monoubiquitin protein levels, which correlated with a shift of ubiquitin mRNAs from larger polysomes to non-translating ribosomes. In contrast, levels of many housekeeping proteins did not change. This apparent reduction in the translation of ubiquitin genes correlated with a modest reduction in protein synthesis rate and formation of large polysomes. To further determine whether Int6 can selectively control translation, we analyzed translation of different 5'-untranslated region reporters and found that indeed, loss of Int6 had differential effects on these reporters. Together the data suggest that Int6 depletion blocks ubiquitin-dependent proteolysis by decreasing both ubiquitin levels and the assembly of functional proteasome machinery, leading to accumulation of oncoproteins, such as SRC3 that can transform mammary epithelium. Our data also raise the possibility that Int6 can further fine-tune protein levels by selectively controlling translation of specific mRNAs.

Sperm storage in the vertebrate female reproductive tract: how does it work so well?

The capacity for sperm storage within the female reproductive tract occurs widely across all groups of vertebrate species and is exceptionally well developed in some reptiles (maximum duration, 7 yr) and fish (maximum duration, >1 yr). Amphibians (most salamanders and one species of frog; duration approximately 5 mo), all birds examined to date and some bats, have also evolved the ability to store spermatozoa in the female reproductive tract. Although there are many reports on both the occurrence of female sperm storage and its adaptive benefits, few studies have been directed toward explaining the mechanisms involved. Phylogenetic evidence suggests that the capacity for sperm storage has evolved independently within different taxonomic groups, and it is by no means clear whether these groups have established similar or different mechanisms or whether simple and common principles have been exploited during evolution. If the process has indeed developed by the invention of numerous different and species-specific mechanisms, it is surprising that none have yet been elucidated by technologists wishing to improve the long-term storage of fresh semen. On the other hand, if there is a simple and common solution to the problem, readily accessed by diverse groups of species, it is equally logical to suppose that the mechanism should be easily discovered in the laboratory. While recognizing that studies on wild species are usually neither practically or ethically easy to undertake, it is clear that there is a huge and largely unexplored field to be investigated.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro.

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39 degrees C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

Artificial insemination for the propagation of CANDES: the reality!

Conservation is about protecting and nurturing species so that they can survive, not only now, but also into the future. Ideally this means protecting genetically diverse populations and not simply breeding a few individuals. Unfortunately, this point is often overlooked by reproductive technologists, especially if they are more accustomed to working with humans, companion animals or agricultural species, where the goals are more usually directed towards obtaining offspring from particular individuals. This approach has tended to antagonise the conservation community, who are quick to develop an unreasonable suspicion of technological solutions, partly because they are unfamiliar with the scientific principles that underpin the reproductive technology. Unfortunately, this mutual failure to recognise that all parties are actually well meaning, has led to separate cultures that barely communicate with each other and thus fail to capitalise on the potential benefits that would come from a good working relationship. Notable successes with reproductive technology have only emerged where such relationships have been forged. In this review, we highlight, mainly for the benefit of the technologist community, the need to foster good working relationships with conservation managers and to recognise that the latest hi-tech approach to animal breeding is more likely to engender suspicion than enthusiasm.

Direct contact between boar spermatozoa and porcine oviductal epithelial cell (OEC) cultures is needed for optimal sperm survival in vitro.

Oviductal epithelial cell (OEC) co-culture prolongs sperm viability and motility in vitro in a number of species including humans and horses. This study has sought to determine the effects of homologous OEC co-culture on boar sperm function. To determine whether the effects on spermatozoa were specifically caused by co-culture with or by OEC secretions, or by both factors together, a number of co-culture and cell-conditioned medium (CM) experiments were conducted. Firstly, Percoll-washed spermatozoa were co-cultured with OECs and pig kidney epithelial (LLC-PK1) cells, and in medium without cells. Secondly, Percoll-washed spermatozoa were incubated with CM derived from both OECs and LLC-PK1 cells and in unconditioned medium. A number of sperm function parameters were assessed after 5, 30, 60, 90, 120, and 180 min, and 24h of co-culturing or incubation with CM. Of all the sperm function parameters investigated, the percentage (%) viability data yielded the most interesting results. OECs (mean+/-S.E.M.; 31.2+/-1.10) were better than LLC-PK1 cells (24.3+/-0.93) at prolonging the viability of unbound spermatozoa after 24h of co-culturing (P<0.05). Also after 24h, the viability of spermatozoa bound to the OECs (77.6+/-1.83) was significantly higher than in the case of the LLC-PK1 cells (53.5+/-1.43; P<0.001). Other sperm function parameters, e.g., capacitation and motility, were also influenced by OEC co-culturing and incubation with CM, although to a lesser degree. In conclusion, porcine homologous OEC co-culture and CM incubation specifically affect sperm function. However, we propose that it is OEC co-culturing, rather than OEC-CM, that has the greater influence.

Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells.

Following insemination, ram spermatozoa bind to oviducal epithelial cells (OEC) in vivo and remain viable for several hours before fertilisation. In the present study, we investigated whether OEC monolayers reproduce this effect in vitro, performing an analysis of ram sperm binding and survival over an extended (48 h) period at 39 degrees C. We wanted to determine whether the reproductive cycle phase and/or oviducal region would influence ram sperm binding and survival in coculture with OEC and whether reproductive and non-reproductive epithelial cells bound and maintained the viability of ram spermatozoa equivalently. Oviducts were separated into groups based on their ovarian state (follicular or luteal) and then divided into two parts (isthmus and ampulla) for OEC isolation. Sheep kidney epithelial cells (Madin-Darby ovine kidney; MDOK) were purchased commercially. Reproductive cycle phase, but not oviducal region, affected sperm binding to OEC. Although more spermatozoa bound to luteal OEC than to follicular OEC at 1 h, at 24 h follicular OEC had bound more spermatozoa than luteal OEC. Generally, spermatozoa that were bound to OEC and MDOK had enhanced viability at each of the time points investigated (1, 6, 24 and 48 h), but the viability of the OEC-bound spermatozoa was greater than that of the MDOK-bound spermatozoa at 48 h. In conclusion, ram sperm-epithelial cell interactions are temporal, dynamic and depend on the origin of the epithelial cells.

CVB translation: lessons from the polioviruses.

Our understanding of coxsackie B virus translation and replication has benefited greatly from half a century of research on the closely related polioviruses. Like poliovirus, coxsackievirus gene expression is controlled largely at the translation level and coxsackievirus infection results in profound changes in the profile of mRNAs with access to the protein synthesis machinery of the host cell. This review chronicles the advances in understanding translational control by the enteroviruses, primarily in poliovirus and clarified by related viruses, and highlights areas where coxsackievirus conforms to or differs from the aggregate model. Basic IRES structure and function, proteins involved in cap-dependent and viral translation, viral modification of translation factors to achieve host translation shutoff and promotion of viral translation are discussed. The translational bases for neurovirulent phenotypes and tissue specificity are also addressed.

Haematoma of anterior ethmoidal artery after reduction of fracture of zygomatic complex.

Extraconal intraorbital haemorrhage is a rare complication after reduction of a fracture of the zygomatic complex. We present a case of postoperative intraorbital haematoma that arose from the anterior ethmoidal artery. We stress the advantages of imaging before decompression and of the medial approach.

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.

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage.

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.

Identification of caspase 3-mediated cleavage and functional alteration of eukaryotic initiation factor 2alpha in apoptosis.

Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.

Cleavage of eukaryotic translation initiation factor 4GII correlates with translation inhibition during apoptosis.

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.

Osseointegration of titanium metal implants in erbium-YAG laser-prepared bone.

Titanium screws were implanted in rat calvarial defects of identical size using either a laser or bur. The aims of this study were to determine whether the screws were able to osseointegrate in a laser-prepared bone defect and to compare the pattern of bone healing around these screws. The optimal laser settings to produce a 0.7-mm-diameter hole in the rat calvaria were determined. A 0.7-mm-diameter hole was prepared on the left calvaria with the erbium-YAG laser to receive a 1-mm-diameter self-threading titanium screw. Each animal also received a 0.7-mm-diameter hole prepared on the right calvaria with a conventional metal bur, and a 1-mm-diameter self-threading screw implant was placed. Rats were killed humanely either 3 weeks or 3 months after surgery, and the skulls were processed in paraffin wax for histological analysis. Laser-prepared defects: At 3 weeks, the screw was surrounded by vital woven bone. The dura mater was perforated, and cystic change was present in the underlying brain tissue. There was active bone formation adjacent to the screw surface, deposited on a thin zone of necrotic bone. At 3 months, the screws were osseointegrated, and the brain tissue was healed by gliosis. Bur-prepared defects: At 3 weeks, there was extensive remodeling around the prepared defect. The dura mater was intact, and there was no damage to the underlying brain. At 3 months, the screws were successfully osseointegrated with bone adjacent to the screw. Osseointegration of titanium screws can be achieved using an erbium-YAG laser to prepare the implant bed.

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.

Muscle-sparing minithoracotomy with intercostal nerve cryoanalgesia: an improved method for major lung resections.

To decrease incisional pain, morbidity, and length of hospital stay (LOS) and, hopefully, to reduce costs, most surgical specialties have turned to minimally invasive procedures to access the body cavities during commonly performed operations. Video-assisted thoracic surgery (VATS) has emerged as the standard approach for a number of diagnostic and therapeutic procedures in thoracic surgery. Major lung resections (lobectomy, bilobectomy, and pneumonectomy), however, can be performed through an incision similar in size to the utility or access thoracotomy used in VATS to remove the specimen. The purpose of this study was to compare an oblique muscle-sparing minithoracotomy with intercostal nerve cryoanalgesia with the standard posterolateral thoracotomy incision and VATS to perform major lung resections. Forty consecutive patients with bronchogenic carcinoma, operated on by a single surgeon, were chronologically divided into two groups, each with equivalent age, sex distribution, physiologic parameters, tumor size, and clinical stage. In addition, data were collected from a MEDLINE search of all published studies in which major lung resections were performed via VATS. The first group (group A, n = 20) underwent posterolateral thoracotomy to access the chest cavity, whereas the patients in the second group (group B, n = 20) underwent oblique minithoracotomy with intercostal nerve cryoanalgesia. Group B compared favorably with group A in LOS (P = 0.002), narcotic requirements (P = 0.001), morbidity (P = 0.042), and cost (P = 0.058). Group B also compared favorably with VATS major lung resection published data regarding LOS and morbidity.

Eukaryotic translation initiation factor 4G is targeted for proteolytic cleavage by caspase 3 during inhibition of translation in apoptotic cells.

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.

The predominant elF4G-specific cleavage activity in poliovirus-infected HeLa cells is distinct from 2A protease.

Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2Apro) is sufficient to cause host translation shutoff through cleavage of elF4G (formerly p220, elF4 gamma) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant elF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2Apro by size exclusion chromatography. 2Apro separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2Apro fractions did not cleave elF4G or an elF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions. Conversely, fractions which contained peak elF4G cleavage activity and only trace amounts of 2Apro efficiently cleaved a peptide substrate derived from the previously mapped elF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant elF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2Apro or its precursors. Quantitation of 2Apro from lysates of infected cells showed that during infections in HeLa cells, 2Apro does not reach molar excess over elF4G, as previously shown to be required for direct elF4G cleavage in vitro. Further, infection of HeLa cells in the presence of 2 mM guanidine-HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2Apro and its precursor 2ABC below detectable levels but was unable to delay the onset of elF4G proteolysis in vivo. The elF4G cleavage activity was still easily detectable in in vitro assays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of elF4G in vivo. Although it was not directly measurable here, 2Apro likely does cleave a portion of elF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2Apro constitutes the bulk of the elF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient elF4G cleavage activity observed in vivo.