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.     

Translational elongation rate changes in encephalomyocarditis virus-infected and interferon-treated cells.

Infection of mouse L cells with encephalomyocarditis virus results in a rapid inhibition of host protein synthesis before the synthesis of viral proteins. Although no alterations in initiation factor activities have been demonstrated in encephalomyocarditis virus-infected mouse cells, a defect in polypeptide chain elongation has been shown to occur in infected cell extracts. We investigated the significance of this elongation defect in the host shutoff phenomenon in vivo. Average polypeptide chain elongation rates were measured at various times after infection. Interferon was used as a reagent to separate temporarily the virus-induced alterations. Encephalomyocarditis virus infection of L cells was shown to lead to a progressive reduction in the elongation rate. Whereas interferon pretreatment delayed the decrease in elongation rate in a dose-dependent manner, it failed to alter the kinetics of host shutoff, suggesting that slowing of elongation steps played no significant role in this phenomenon. In addition, interferon pretreatment of either mock-infected or virus-infected cells led to no elongation defect that could be attributed to interferon action.     

Poliovirus translation: a paradigm for a novel initiation mechanism

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Autophagy promotes the replication of encephalomyocarditis virus in host cells

A growing number of studies have demonstrated that autophagy has a diverse role in the infection process of different pathogens. However, to date, it is unknown whether autophagy is activated in encephalomyocarditis virus (EMCV)-infected host cells, and if so, what its role is in this process. In the present study, we first demonstrated that EMCV infection significantly increases the number of double- and single-membrane vesicles in the cytoplasm of host cells. It was then confirmed that these observed vesicles are indeed related to autophagy, and that EMCV replication is required for the induction of autophagy by examining LC3-I/-II conversion and p62/SQSTM1 degradation using immunoblotting. Next, we performed confocal immunofluorescence analysis and discovered that, during EMCV replication, both the nonstructural protein 3A and capsid protein VP1 colocalized with LC3. The colocalizations of both 3A and VP1 protein with autophagosome-like vesicles were further confirmed using immunoelectron microscopy, indicating that EMCV undergoes RNA replication on the membranes of these vesicles. Finally, we used pharmacological regulators and siRNAs to examine the role of autophagy in EMCV replication. Our results suggest that autophagy not only promotes the replication of EMCV in host cells, but it also provides a topological mechanism for releasing cytoplasmic viruses in a nonlytic manner. Noticeably, the autophagic pharmaceuticals we used had no significant effect on virus entry or cell viability, both of which may affect viral replication. To our knowledge, ours is the first strong evidence indicating that autophagy is involved in EMCV infection in host cells.     

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.     

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3''-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.     

Adenovirus VAI RNA facilitates the initiation of translation in virus-infected cells

The adenovirus VAI RNA is a small polymerase III-transcribed species that is required for optimal translation of mRNAs late after infection. Mutant dl331 fails to produce this RNA species and, as a result, grows poorly. Mutant-infected cells contain normal levels of late mRNAs, but reduced levels of polypeptides are synthesized late after infection. Translational elongation occurs at normal rates in mutant, as compared to wild-type, virus-infected cells. Initiation of translation occurs with reduced efficiency in dl331 -infected cells. VAI RNA is required for formation of a stable 48S preinitiation complex and very likely functions to facilitate the interaction between 43S preinitiation complex and mRNA to form the 48S species.     

Activating translation by phase separation: a novel mechanism for driving spermiogenesis

Science China Life Sciences -     

Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism

Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

Inhibition of DNA synthesis in varicella-zoster virus-infected cells by BV-araU.

The inhibitory effect of BV-araU on DNA synthesis in human embryonic lung cells infected with varicella-zoster virus (VZV) or herpes simplex virus type 1 (HSV-1) was compared with that of acyclovir. Cellular uptake of [3H]thymidine and its incorporation into DNA was markedly stimulated by the infection with VZV or HSV-1, suggesting that the incorporation was mainly due to viral DNA synthesis. DNA synthesis in VZV-infected cells was dose-dependently suppressed by BV-araU and acyclovir, although cellular uptake of [3H]thymidine decreased in cells treated with a high concentration of drugs for an extended time. DNA synthesis in HSV-1-infected cells was also markedly inhibited by both drugs in a dose-dependent manner, without affecting cellular uptake of [3H]thymidine. The concentration of drugs inhibiting DNA synthesis was well correlated to their in vitro anti-VZV and anti-HSV-1 activities. The inhibitory concentration of BV-araU for DNA synthesis in VZV-infected cells was one-thousandth of that of acyclovir. Our results suggest that the antiviral action of BV-araU against VZV and HSV-1 is based on the inhibition of DNA synthesis in herpesvirus-infected cells.     

Comparison of initiation rates of encephalomyocarditis virus and host protein synthesis in infected cells.

The relative initiation rates for encephalomyocarditis virus mRNA and host mRNA's in infected cells were measured using two independent techniques. In both cases the results showed that viral mRNA initiates at a much higher rate than host mRNA'S. This difference was observed midway in the infectious cycle, well before virus-induced cytopathic effects (leakage of low-molecular-weight metabolites, failure to exclude trypan blue) were apparent. These results confirm that encephalomyocarditis viral mRNA is a more efficient initiator than host mRNA's in vivo, as has previously been demonstrated in in vitro experiments.