Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.     

Mengovirus leader is involved in the inhibition of host cell protein synthesis.

The presence of a leader peptide in picornaviruses is restricted to the Cardiovirus and Aphthovirus genera. However, the leader peptides of these two genera are structurally and functionally unrelated. The aphthovirus leader is a protease involved in viral polyprotein processing and host cell translation shutoff. The function of the cardiovirus leader peptide is still unknown. To gain an insight into the function of the cardiovirus leader peptide, a mengovirus leader peptide deletion mutant was constructed. The deletion mutant was able to grow at a reduced rate in baby hamster kidney cells (BHK-21). Mutant virus production in mouse fibroblasts (L929 cells), however, could be demonstrated only after inoculation of BHK-21 cells with the transfected L929 cells. Analysis of cellular and viral protein synthesis in mutant virus-infected cells showed a delayed inhibition of host cell protein synthesis and a reduced production of viral proteins. In a single-cycle infection, mutant virus produced only 1% of wild-type virus yield at 8 h postinfection. Host cell translation shutoff in L929 cells infected with mutant virus was restored by the addition of the kinase inhibitor 2-aminopurine. Mutant virus production in 2-aminopurine-treated L929 cells was increased to 60% of wild-type virus yield at 8 h postinfection. Our results suggest that the cardiovirus leader peptide is involved in the inhibition of host cell protein synthesis.     

Rapamycin stimulates viral protein synthesis and augments the shutoff of host protein synthesis upon picornavirus infection.

The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not cap-independent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus.     

Rapamycin and Wortmannin Enhance Replication of a Defective Encephalomyocarditis Virus

Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)–FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation and activation of the suppressor of cap-dependent translation, 4E-BP1. Encephalomyocarditis virus (EMCV) infection leads to activation of 4E-BP1 at the time of host translation shutoff. Consistent with these data, rapamycin mildly enhances the synthesis of viral proteins and the shutoff of host cell protein synthesis after EMCV infection. In this study, two defective EMCV strains were generated by deleting portions of the 2A coding region of an infectious cDNA clone. These deletions dramatically decreased the efficiency of viral protein synthesis and abolished the virus-induced shutoff of host translation after infection of BHK-21 cells. Both translation and processing of the P1-2A capsid precursor polypeptide are impaired by the deletions in 2A. The translation and yield of mutant viruses were increased significantly by the presence of rapamycin and wortmannin during infection. Thus, inhibition of the PI3 kinase-FRAP signaling pathway partly complements mutations in 2A protein and reverses a slow-virus phenotype.     

Host range restriction of vaccinia virus in Chinese hamster ovary cells: relationship to shutoff of protein synthesis.

Chinese hamster ovary cells were found to be nonpermissive for vaccinia virus. Although early virus-induced events occurred in these cells (RNA and polypeptide synthesis), subsequent events appeared to be prevented by a very rapid and nonselective shutoff of protein synthesis. Within less than 2 h after infection, both host and viral protein syntheses were arrested. At low multiplicities of infection, inhibition of RNA synthesis with cordycepin resulted in failure of the virus to block protein synthesis. Moreover, infection of the cells in the presence of cycloheximide prevented the immediate onset of shutoff after reversal of cycloheximide. Inactivation of virus particles by UV irradiation also impaired the capacity of the virus to inhibit protein synthesis. These results suggested that an early vaccinia virus-coded product was implicated in the shutoff of protein synthesis. Either the nonpermissive Chinese hamster ovary cells were more sensitive to this inhibition than permissive cells, or a regulatory control of the vaccinia shutoff function was defective.     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

Effects of anti-E2 monoclonal antibody on sindbis virus replication in AT3 cells expressing bcl-2.

Antibodies directed to Sindbis virus (SV) envelope protein E2 are able to control virus replication in vivo and in persistently infected cultures of neurons in vitro. We investigated the mechanisms by which anti-E2 monoclonal antibody (MAb) alters virus replication by using AT3 rat prostatic carcinoma cells expressing the inhibitor of apoptosis bcl-2. Treatment of SV-infected AT3-bcl-2 cells with anti-E2 MAb G5 for 2 h decreased the rate of virus release for 6 to 8 h after removal of the antibody. Electron microscopic analysis of MAb-treated cells revealed that failure of virus release was linked to a defect in the budding process. The decrease in extracellular virus particles occurred despite continued formation of nucleocapsids and synthesis of envelope glycoproteins. MAb treatment delayed the inhibition of K+ influx and shutoff of host cell protein synthesis by SV infection in a dose-dependent manner. Synthesis of host cell factors and of nonstructural polyprotein precursors required for the formation of initial replication complexes was also prolonged, causing a slower shutdown of overall viral RNA synthesis. We conclude that one mechanism by which anti-E2 MAb treatment down-regulates SV replication is by reestablishing certain critical host cell functions in infected cells.     

Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.     

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR^?/? mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.     

The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation.

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.     

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.     

Shutoff of HeLa cell protein synthesis by encephalomyocarditis virus and poliovirus: a comparative study.

Previous experimental results have suggested that poliovirus and encephalomyocarditis (EMC) virus employ very different mechanisms for shutting off host protein synthesis. However, this conclusion is suspect, inasmuch as different cell types were used for the two viruses; hence the apparent mechanistic differences might be specific for cell type and not virus type. To test this possibility we compared shutoff mechanisms in poliovirus- and EMC virus-infected HeLa cells. Striking differences were seen: poliovirus-induced shutoff was much more rapid and extensive than that induced by EMC virus; relative translation rates of certain host proteins were inhibited to different extents by the two viruses; initiation factors prepared from poliovirus-infected cells were specifically defective for translation of capped mRNA's in vitro, whereas those from EMC virus-infected cells were not. These results indicate that EMC virus and poliovirus employ different mechanisms for the shutoff of HeLa cell protein synthesis. This conclusion is consistent with much earlier work and indicates that many differences previously reported are specific to virus type.