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.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection

For many members of the Picornaviridae family, infection of cells results in a shutoff of host protein synthesis. For rhinoviruses and enteroviruses, the shutoff has been explained in part by the cleavage of eukaryotic initiation factor 4GI (eIF4GI), a component of the cap-binding protein complex eIF4F. The cleavage of eIF4GI is mediated by the virus-specific proteinase 2Apro and results in inhibition of cap-dependent, but not cap-independent, translation. The inhibition of host protein synthesis after infection with human rhinovirus 14 (HRV-14) lags behind the cleavage of eIF4GI. Recently, we discovered a functional homolog of eIF4GI, termed eIF4GII, and showed that cleavage of eIF4GII coincides with the shutoff of host cell protein synthesis after poliovirus infection (Gradi et al., Proc. Natl. Acad. Sci. USA 95:11089-11094, 1998). We wished to determine whether eIF4GII cleavage kinetics could also explain the lack of correlation between the kinetics of eIF4GI cleavage and the shutoff of host protein synthesis after rhinovirus infection. In this study, we examined the correlation between human rhinovirus-induced shutoff of host protein synthesis and cleavage of eIF4GI and eIF4GII. In HRV-14-infected HeLa cells, almost no intact eIF4GI could be detected by 4 h postinfection, while only 4% of eIF4GII was cleaved at this time. By 6 h, however, 67% of eIF4GII was cleaved, and this cleavage coincided with a significant (60%) decline of host translation. These results suggest that cleavage of both eIF4GI and eIF4GII is required for HRV-mediated inhibition of host cell protein synthesis and that the cleavage of eIF4GII is the rate-limiting step in the shutoff of host cell protein synthesis after rhinovirus infection.     

The host protein required for in vitro replication of poliovirus is a protein kinase that phosphorylates eukaryotic initiation factor-2

The HeLa cell protein (host factor) required for in vitro replication of poliovirus has been identified as a 67,000 dalton phosphoprotein. The purified protein displays three activities in vitro: stimulation of poliovirus RNA synthesis in the presence of poliovirus replicase, apparent self-phosphorylation, and phosphorylation of the alpha-subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2). All three activities can be removed or inhibited by an antibody to host factor. Partially purified preparations of reticulocyte eIF-2 contain a similar phosphoprotein and display host factor activity in the viral RNA synthesis assay in vitro. In vitro phosphorylation of the 67 kd protein can be stimulated by low concentrations of double-stranded RNA. Addition of phosphorylated host factor in an in vitro RNA synthesis assay significantly changes the kinetics of viral RNA synthesis, indicating that protein phosphorylation may play an important role in viral RNA replication.     

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.     

Toxoplasma gondii: Growth in the absence of host cell protein synthesis

Host cell protein synthesis continues when cultured cells are infected by Toxoplasma gondii. In order to determine if this host function is necessary for the parasite we used two independent methods that specifically block cellular protein synthesis. In the first, we infected a temperature-sensitive Chinese hamster ovary cell mutant that has a thermolabile leucyl tRNA synthetase. At the restrictive temperature of 40 C, the mutant cells showed only negligible protein synthesis that was probably mitochondrial. At this temperature, the growth and nucleic acid synthesis of T. gondii proceeded normally and [³H]leucine was specifically incorporated into the parasite as demonstrated by autoradiography. A secpnd method for blocking protein synthesis by the host cell employed treatment of uninfected human fibroblast cells with muconomycin A, an inhibitor of initiation. Repeated washing of monolayer cultures reduced the free muconomycin A to an insignificant level while the cells remained incapable of protein synthesis. T. gondii infected and grew normally in the inhibited cells. Autoradiographic localization of the incorporation of [³H]leucine showed that it was almost exclusively in the intracellular parasites in the cells pretreated with muconomycin A. In the untreated control most of the [³H]leucine was incorporated by the host cell rather than the parasite. We conclude that de novo protein synthesis by the host cell is not required to support the growth of intracellular T. gondii.     

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single‐cell level are still lacking. To address this need, we apply O‐propargyl‐puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single‐cell level. In conclusion, OPP‐labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.     

Replication of Mengovirus in HeLa Cells Preinfected with Nonreplicating Poliovirus

The replication of mengovirus in HeLa cells preinfected with poliovirus in the presence of 10(-3) M guanidine was investigated. Although host cell protein synthesis is inhibited by the presence of nonreplicating poliovirus, it is found that mengovirus ribonucleic acid (RNA) and protein synthesis proceed normally under the same conditions. Furthermore, no effects on mengovirus growth by poliovirus can be detected either when Mengo protein synthesis is interrupted by Acti-Dione or when its RNA synthesis is reduced by incubation at 28 C. It is suggested that the poliovirus inhibitory factor may be able to distinguish between an RNA element required in the protein-synthesizing apparatus of the host cell and a comparable element in that of the heterologous virus.     

Herpes simplex virus 1 infection alters the mRNA translation processing in L-02 cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off. Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Human Cytomegalovirus Protein pUL117 Targets the Mini-Chromosome Maintenance Complex and Suppresses Cellular DNA Synthesis

Modulation of host DNA synthesis is essential for many viruses to establish productive infections and contributes to viral diseases. Human cytomegalovirus (HCMV), a large DNA virus, blocks host DNA synthesis and deregulates cell cycle progression. We report that pUL117, a viral protein that we recently identified, is required for HCMV to block host DNA synthesis. Mutant viruses in which pUL117 was disrupted, either by frame-shift mutation or by a protein destabilization-based approach, failed to block host DNA synthesis at times after 24 hours post infection in human foreskin fibroblasts. Furthermore, pUL117-deficient virus stimulated quiescent fibroblasts to enter S-phase, demonstrating the intrinsic ability of HCMV to promote host DNA synthesis, which was suppressed by pUL117. We examined key proteins known to be involved in inhibition of host DNA synthesis in HCMV infection, and found that many were unlikely involved in the inhibitory activity of pUL117, including geminin, cyclin A, and viral protein IE2, based on their expression patterns. However, the ability of HCMV to delay the accumulation of the mini-chromosome maintenance (MCM) complex proteins, represented by MCM2 and MCM4, and prevent their loading onto chromatin, was compromised in the absence of pUL117. When expressed alone, pUL117 slowed cell proliferation, delayed DNA synthesis, and inhibited MCM accumulation. Knockdown of MCM proteins by siRNA restored the ability of pUL117-deficient virus to block cellular DNA synthesis. Thus, targeting MCM complex is one mechanism pUL117 employs to help block cellular DNA synthesis during HCMV infection. Our finding substantiates an emerging picture that deregulation of MCM is a conserved strategy for many viruses to prevent host DNA synthesis and helps to elucidate the complex strategy used by a large DNA virus to modulate cellular processes to promote infection and pathogenesis.     

Herpes Simplex Virus 1 Infection Alters the mRNA Translation Processing in L-02 Cells

HSV-1 infection-mediated regulation of mRNA translation in host cells is a systematic and complicated process. Investigation of the details of this mechanism will facilitate understanding of biological variations in the viral replication process and host cells. In this study, a comparative proteomics technology platform was applied by two-dimension electrophoresis of HSV-1 infected normal human L-02 cell and control cell lysates. The observed protein spots were analyzed qualitatively and quantitatively by the PDQuest software package. A number of the different observed protein spots closely associated with cellular protein synthesis were identified by matrix-assisted laser-desorption ionization-time of flight-mass spectrometry （MALDI-TOF-MS）. The expression levels of the RPLP1 protein, which is required for mRNA translation, and KHSRP protein, which is involved in rapid decay of mRNA, were up-regulated, whereas the expression level of RNP H2, which is involved in positive regulation on the mRNA splicing process, was down-regulated. All of these results suggest that HSV-1 infection can influence cellular protein synthesis via modulation of cellular regulatory proteins involved in RNA splicing, translation and decay, resulting in optimisation of viral protein synthesis when cellular protein synthesis is shut off Although there is need for further investigations regarding the detailed mechanisms of cellular protein control, our studies provide new insight into the targeting of varied virus signaling pathways involved in host cellular protein synthesis.     

Mechanism of Mengo Virus-Induced Cell Injury in L Cells: Use of Inhibitors of Protein Synthesis to Dissociate Virus-Specific Events

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.     

Effect of hypertonic conditions on protein synthesis in cells productively infected with simian virus 40.

Hypertonic medium selectively suppressed the synthesis of most host cell polypeptides relative to the synthesis of simian virus 40 capsid polypeptides and a minority of cellular polypeptides, notably histones. Under optimal hypertonic conditions, the synthesis of the major capsid polypeptide (VP1) is enhanced about sevenfold relative to host polypeptide synthesis. Because of the small amounts of the other nonhistone capsid polypeptides (VP2) and VP3) present in cell lysates, it was difficult to quantitate the extent, if any, of their enhancement. The maintenance of the restricted pattern of protein synthesis caused by hypertonic medium was dependent on continual peptide chain initiations. The resistance of viral protein synthesis to hypertonic conditions provides a means of detecting relatively low levels of intracellular viral protein synthesis. Analysis of the specific activity of the acid-soluble [3H]lysine pool indicated that the rate of incorporation of [3H]lysine into protein was an overestimation of the actual rate of overall protein synthesis occurring in cells exposed to hypertonic as compared to isotonic conditions. Since it is likely that both cellular and viral protein synthesis draw lysine from a single pool, this change in pool specific activity does not affect the analysis of relative rates of protein synthesis at a given level of tonicity.     

Human rhinovirus 14 infection of HeLa cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mRNA translation in vitro.

One of the characteristics of picornavirus infection of cells in tissue culture is a specific inhibition of utilization of host cell mRNA for protein synthesis. In this study we show that human rhinovirus 14 is similar to poliovirus in that the inhibition of host cell translation that occurs during infection correlates with the proteolytic cleavage of an Mr 220,000 subunit of the cap-binding protein complex.