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).     

2Apro is a multifunctional protein that regulates the stability, translation and replication of poliovirus RNA

Poliovirus 2A(pro) is required for the inhibition of host cell protein synthesis and efficient viral replication. We investigated the role of 2A(pro) in regulating viral RNA stability, translation and replication in HeLa S10 reactions. The protease activity of 2A(pro) or its polyprotein precursors, 2AB or P2, was required to increase the stability of viral RNA and prolong translation. Since other viral proteins were not required for the observed effects of 2A(pro), it is likely that a cellular protein(s) modified by 2A(pro) mediated these effects on stability and translation. In addition, the protease activity of 2A(pro) stimulated negative-strand initiation by approximately five-fold but had no effect on positive-strand initiation. The 2A(pro) stimulation of negative-strand synthesis was independent of its effect on stability and translation. These findings further extend the previously known functions of protein 2A(pro) to include its role in increasing RNA stability, prolonging translation and stimulating negative-strand synthesis.     

The viral protein 3CD induces an equilibrium between the viral protein and RNA synthesis in a cell-free system for poliovirus replication

Summary.  In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log₁₀. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log₁₀ by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system. Received April 2, 2001 Accepted November 15, 2001     

Foot-and-mouth disease virus infection induces proteolytic cleavage of PTB, eIF3a,b, and PABP RNA-binding proteins

Foot-and-mouth disease virus (FMDV) infection induces major changes in the host cell including the shutoff of cellular protein synthesis. Here, protein extracts from FMDV-infected cells have been used to monitor changes in the profile of RNA-binding factors interacting with regulatory regions of the viral RNA. Relevant differences have been detected in the pattern of interaction with proteins prepared from either infected or uninfected cells with RNA probes encompassing the internal ribosome entry site (IRES), the 5' and 3'end regions. The binding patterns obtained for two divergent FMDV isolates showed differences depending on the viral isolate used. The identity of the host proteins giving a shifted binding pattern to RNA regulatory regions has been inferred by immunoblotting. Our results show that polypyrimidine tract-binding protein (PTB) and two subunits of translation initiation factor eIF3 interacting with the IRES undergo proteolytic processing during FMDV infection. In addition, poly(A)-binding protein (PABP), interacting with the 3'end of the viral RNA is partially processed. Proteolysis of eIF3a, eIF3b, PABP and PTB correlated with the extent of cytopathic effect induced by FMDV in infected cells.     

Differential inhibition of cellular and Sindbis virus translation by brefeldin A

Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As a result, this antibiotic impedes the maturation of viral glycoproteins of enveloped viruses and viral genome replication in several virus species. In the present work, we show that translation of subgenomic mRNA from Sindbis virus, which in contrast to cellular translation is resistant to brefeldin A after prolonged treatment. The phosphorylation of eIF2alpha as a result of brefeldin A treatment correlates with the inhibition of cellular translation, while late viral protein synthesis is resistant to this phosphorylation. The effect of brefeldin A on Sindbis virus replication was also examined using a Sindbis virus replicon. Although brefeldin A delayed viral RNA synthesis, translation by non-replicative viral RNAs was not affected, reinforcing the idea that brefeldin A delays viral RNA replication, but does not directly affect Sindbis virus protein synthesis.     

Inhibition by prostaglandin PGA1 on the multiplication of influenza virus is a dose-dependent effect

Cyclopentenone prostaglandins (PGs), are strong inhibitors of the multiplicative cycle of a wide variety of enveloped RNA and DNA viruses. Their antiviral activity is generally associated with alterations in the synthesis or maturation of specific virus proteins. In this report, we describe the effect of cyclopentenone PGA1 on the replication of influenza A virus Ulster 73 in LLC-MK2 cells. PGA1 was found to inhibit viral replication in a dose-dependent fashion and virus particle yield was reduced at a PGA1 concentration, which did not suppress protein synthesis in mock-infected cells. The kinetic of late viral protein synthesis was delayed in PGA1-treated cells till 10 h post-infection; after that period, viral polypeptide synthesis appeared to be similar in PGA1-treated as well as untreated cells both infected by Ulster 73 virus. This finding suggests that PGA1 might interfere with one or more events in the viral multiplicative cycle such as protein synthesis and assembly, correct insertion of virus polypeptides into the cell membrane and, or maturation of Ulster 73 virion particles. In particular, inhibition of viral replication in LLC-MK2 cells by PGA1 is accompanied by the induction of a cellular polypeptide of 70K molecular weight. We identified this cell protein as a heat shock protein (HSP) related to the inducible isoform of HSP 70, a polypeptide of 72K molecular weight. Induction of this polypeptide by PGA1 was found to be dose-dependent and a substantial accumulation could be seen at a PGA1 concentration that did not inhibit cell protein synthesis in uninfected cells. HSP 70 synthesis started after the beginning of PGA1 treatment and remained at the same level for at least 10 h, leading us to hypothesize that the delay of production of late Ulster 73 proteins could be the consequence of HSP 70 synthesis. These results suggest that HSP 70 could play a role in the antiviral activity of cyclopentenone PGA1 in LLC-MK2 cells.     

Induction of host cell protein synthesis by human herpesvirus 6.

We observed an increase in host cell protein synthesis in human cord blood lymphocytes (CBL) infected with human herpesvirus 6 relative to uninfected cultures. The magnitude of this effect could not be explained by a smaller decrease in cell number in the infected cultures. The induction of host cell protein synthesis by HHV-6 does not appear to be mediated by a stable soluble factor present in the infected cell culture supernatant. When CBL were infected with virus that had been exposed to ultraviolet irradiation (UV) for various intervals, we found that the level of increase in cell number, host protein synthesis, viral DNA and viral antigen was inversely proportional to the length of time of virus exposure to UV. No increase in cell number or host cell protein synthesis was seen in CBL infected in the presence of 50 micrograms/ml phosphonoacetic acid, an inhibitor of HHV-6 DNA replication. These results indicate that components of input virions do not induce the increased protein synthesis and that the induction is dependent on viral DNA replication.     

Mechanism of the interferon alpha response against hepatitis C virus replicons

Interferon alpha (IFN-alpha) inhibits hepatitis C virus (HCV) replication in vivo and in cell cultures by one or several mechanisms that are not yet understood. We sought to identify the viral targets of the IFN-alpha-induced cellular antiviral program in Huh7 cells expressing HCV subgenomic replicons. Our results revealed a tight linkage between translation, assembly of replication complexes and viral RNA synthesis, and indicated that the stability of amplified plus strand RNA was reduced in the presence of the cytokine. Moreover, HCV internal ribosomal entry site (IRES)-directed translation was inhibited approximately 2-fold in IFN-treated cells. In contrast, the synthesis of viral RNA did not seem to be directly affected by the antiviral program induced by the cytokine. Our results were consistent with a model predicting that the IFN-alpha-induced antiviral program could inhibit multiple steps of the HCV replication cycle, leading to a reduction in viral protein synthesis and eventually inhibition of viral RNA amplification.     

Synthesis of VSV RNPs in vitro by cellular VSV RNPs added to uninfected HeLa cell extracts: VSV protein requirements for replication in vitro

Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited.     

Protein synthesis in vaccinia virus-infected cells

Summary Human Hep-2 cells were submitted to hypertonic shock (210 mM NaCl) to block host protein synthesis before infection with vaccinia virus. With the start of infection, the medium isotonicity (116 mM NaCl) was restored, and the effect of viral infection on the recovery of host polyribosomes and protein synthesis was studied. Although host translation blockage was released together with infection, vaccinia virus did not affect immediately host protein synthesis. During the first hour of recovery, infected cells could perfectly rebuild the polyribosome profile and recuperate the rate of protein synthesis. Also, during recovery, formation of the initiation complex for protein synthesis was not affected by viral infection. In this period, viral mRNA and proteins were detected by slot blot and SDS-polyacrylamide gel electrophoresis. The inhibitory effect of vaccinia virus on host translation was observed after the second hour of infection. These findings suggest that vaccinia virus-mediated shutoff occurs in a later period during infection, in parallel with viral mRNA accumulation in the polyribosomes and after the on-set of viral DNA replication.     

Neutralizing innate host defenses to control viral translation in HSV-1 infected cells

Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its alpha subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2alpha. The gamma(1)34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1alpha and regenerates pools of active eIF2 by removing the inhibitory phosphate from the alpha subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2alpha kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1-infected cells.     

Effect of ribosome-inactivating proteins on virus-infected cells. Inhibition of virus multiplication and of protein synthesis

Summary HEp-2 cells were infected with herpes simplex virus-1 (HSV-1) or with poliovirus I in the presence of plant proteins which inactivate ribosomes in cell-free systems, while exerting scarce effect on whole cells. Ribosome-inactivating proteins used were gelonin, from the seeds ofGelonium multiflorum, an inhibitor from the seeds ofMomordica charantia, dianthin 32, from the leaves ofDianthus caryophyllus (carnation), and PAP-S, from the seeds ofPhytolacca americana (pokeweed). All proteins tested had the following effects: 1. They reduced viral yield; 2. They decreased HSV-1 plaque-forming efficiency; 3. They inhibited protein synthesis more in infected than in uninfected cells. These results strongly suggest that ribosome-inactivating proteins impair viral replication by inhibiting protein synthesis in virus-infected cells, in which presumably they enter more easily than in uninfected cells.With 3 Figures     

NEUTRALIZING INNATE HOST DEFENSES TO CONTROL VIRAL TRANSLATION IN HSV-1 INFECTED CELLS

Lytic replication of many viruses activates an innate host response designed to prevent the completion of the viral lifecycle, thus impeding the spread of the infection. One branch of the host's complex reaction functions to incapacitate the cellular translational machinery on which the synthesis of viral polypeptides completely depends. This is achieved through the activation of specific protein kinases that phosphorylate eIF2 on its α subunit and inactivate this critical translation initiation factor. However, as continued synthesis of viral proteins is required to assemble the viral progeny necessary to transmit the infection to neighboring cells, viruses have developed a variety of strategies to counter this cellular response. Genetic and biochemical studies with herpes simplex virus type 1 (HSV-1) have revealed that the virus produces at least two discrete products at different times during its replicative program that act to prevent the accumulation of phosphorylated eIF2α. The γ₁34.5 gene product is expressed first, encoding a regulatory subunit that binds the cellular protein phosphatase 1α and regenerates pools of active eIF2 by removing the inhibitory phosphate from the α subunit. The second function, encoded by the product of the Us11 gene, specifies a double-stranded RNA-binding protein that prevents activation of PKR, a cellular eIF2α kinase. Together, both proteins cooperate to overcome the antiviral response of the host and properly regulate translation in HSV-1–infected cells.     

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (−)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (−)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (−)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.     

Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

Interaction between zucchini yellow mosaic potyvirus RNA-dependent RNA polymerase and host poly-(A) binding protein

Viral replication depends on compatible interactions between a virus and its host. For RNA viruses, the viral replicases (RNA-dependent RNA polymerases; RdRps) often associate with components of the host translational apparatus. To date, host factors interacting with potyvirus replicases have not been identified. The Potyviridae, which form the largest and most economically important plant virus family, have numerous similarities with the animal virus family, the Picornaviridae. Potyviruses have a single-stranded, plus sense genome; replication initiates at the viral-encoded, 3' poly-(A) terminus. The yeast two-hybrid system was used to identify host plant proteins associating with the RdRp of zucchini yellow mosaic potyvirus (ZYMV). Several cDNA clones representing a single copy of a poly-(A) binding protein (PABP) gene were isolated from a cucumber (Cucumis sativus L.) leaf cDNA library. Deletion analysis indicated that the C-terminus of the PABP is necessary and sufficient for interaction with the RdRp. Full-length cucumber PABP cDNA was obtained using 5' RACE; in vitro and Escherichia coli-expressed PABP bound to poly-(A)-Sepharose and ZYMY RdRp with or without the presence of poly-(A). This is the first report of an interaction between a viral replicase and PABP and may implicate a role for host PABP in the potyviral infection process.     

Replication of potato virus X RNA is altered in coinfections with potato virus Y

Potato virus X (PVX) and potato virus Y (PVY) may coinfect tobacco to cause a classic synergistic disease. In the acute stage the disease is characterized by a dramatic increase in the accumulation of infectious PVX particles, with no corresponding increase or decrease in the accumulation of PVY. The accumulation of PVX genomic RNA and coat protein has been examined in doubly versus singly infected tobacco leaves. These experiments indicate that the levels of both viral components increase in doubly infected plants to about the same extent as the level of infectious PVX particles. The level of PVX subgenomic coat protein mRNA found associated with polyribosomes of synergistically infected plants is also increased to a similar extent. Pulse labelling experiments suggest that the increase in PVX coat protein is due to an increased rate of synthesis. The level of PVX (-) strand RNA template increases disproportionately in doubly infected tissue, to a level three times higher than that of the virion or its component parts. This result suggests that PVX/PVY synergism involves an alteration in the normal regulation of the relative levels of PVX (+) and (-) strand RNAs during viral replication.