Protein Synthesis In Virus-infected Cells Research Articles

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus.

Alternative ferritin mRNA translation via internal initiation

Ferritin stores and detoxifies an excess of intracellular iron, and thereby plays an important role in the metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival, and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. In light of the well-established transcriptional induction of ferritin by inflammatory signals, we examined how ferritin mRNA translation responds to stress conditions. We first used HT1080 fibrosarcoma cells engineered for coumermycin-inducible expression of PKR, a stress kinase that inhibits protein synthesis in virus-infected cells by phosphorylating eIF2α. In this setting, iron triggered partial ferritin mRNA translation despite a PKR-induced global shutdown in protein synthesis. Moreover, iron-mediated ferritin synthesis was evident in cells infected with an attenuated strain of poliovirus; when eIF4GI was cleaved, eIF2α was phosphorylated, and host protein synthesis was inhibited. Under global inhibition of protein synthesis or specific inhibition of ferritin mRNA translation in cells overexpressing PKR or IRP1, respectively, we demonstrate association of ferritin mRNA with heavy polysomes. Further experiments revealed that the 5' untranslated region (5' UTR) of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES). Our data are consistent with the existence of an alternative, noncanonical mechanism for ferritin mRNA translation, which may primarily operate under stress conditions to protect cells from oxidative stress.

Magnesium-Dependent Interaction of PKR with Adenovirus VAI

Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the α-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade the antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg 2+. Two PKR monomers bind in the absence of Mg 2+, but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

A New Cistron in the Murine Hepatitis Virus Replicase Gene

We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.

Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus-Infected Cells

A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production. One of the common viral targets is the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha). In this report, we show that phosphorylation of eIF-2alpha was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus (IBV). To understand whether this suppression is through inhibition of protein kinase R (PKR), the double-stranded-RNA-dependent kinase that is one of the main kinases responsible for phosphorylation of eIF-2alpha, cells infected with IBV were analyzed by Western blotting. The results showed that the level of phosphorylated PKR was greatly reduced in IBV-infected cells. Overexpression of IBV structural and nonstructural proteins (nsp) demonstrated that nsp2 is a weak PKR antagonist. Furthermore, GADD34, a component of the protein phosphatase 1 (PP1) complex, which dephosphorylates eIF-2alpha, was significantly induced in IBV-infected cells. Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34, eIF-2alpha, and PKR, as well as their mutant constructs in virus-infected cells, showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication. Taken together, these results confirm that IBV has developed a combination of two mechanisms, i.e., blocking PKR activation and inducing GADD34 expression, to maintain de novo protein synthesis in IBV-infected cells and, meanwhile, to enhance viral replication.

Inhibition of host protein synthesis in B95a cells infected with the HL strain of measles virus

The shut-off of host protein synthesis in virus-infected cells is one of the important mechanisms for viral replication. In this report, we showed that the HL strain of measles virus (MeV-HL) as well as other field isolates, which were isolated from human blood lymphocytes using B95a cells, induce the shut-off in B95a cells. Since the Edmonston strain of MeV failed to induce the shut-off in B95a cells, the ability to induce the shut-off was considered to be dependent on virus strains. Although, the modification of eukaryotic translation initiation factors (eIF) including eIF4G, eIF4E, and 4E-BP1 was reported for shut-off by various viruses, the involvement of these eIFs was not observed in MeV-HL-infected B95a cells. Instead, the accumulation of phosphorylated eIF2alpha was found to coincide to the decrease of host protein synthesis, suggesting the involvement of phosphorylation of eIF2alpha in inhibition of translation as one of the mechanisms of the shut-off.

Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR.

The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2alpha to downregulate protein synthesis in virus-infected cells. Two double-stranded RNA binding domains (dsRBDs) in the N-terminal half of PKR are thought to bind the activator double-stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization domains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein increased eIF2alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double-stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.

Reovirus sigma 3 protein: dsRNA binding and inhibition of RNA-activated protein kinase.

The interferon-inducible, double-stranded (ds)RNA-activated protein kinase PKR is clearly an important mediator of interferon action and has also been implicated in the induction of apoptosis (Lee and Esteban 1994; Kibler et al. 1997) and in the inhibition of cellular protein synthesis in virus-infected cells. Much of the compelling evidence for the role of PKR in virus replication has come from the study of viruses such as reovirus that encode inhibitors of PKR. This review will discuss the genetic and biochemical data suggesting that reovirus σ3 protein, acting as a dsRNA-binding protein, functions as an inhibitor of PKR.

Antiviral Agents of Plant Origin. Antiherpetic Activity of Acacetin

The effect of acacetin isolated from Scoparia dulcis and several related flavonoids on herpes simplex virus type 1 (HSV-1) was studied in vitro by the method of plaque yield reduction. Among these compounds, acacetin was shown to be the most potent agent and caused dose-dependent inhibition of virus replication. Acacetin had a weak virucidal activity at higher concentrations. Analysis of early events following infection showed that attachment of the virus to host cells and penetration were unaffected by acacetin. Acacetin was found to exert strong inhibition of protein synthesis in virus-infected cells but not in uninfected cells. The transcription of immediate-early genes and translation of their transcripts were in particular almost stopped by acacetin even at a lower concentration. These selective effects can be attributed mainly to the antiviral activity of acacetin.

Yucca leaf protein (YLP) stops the protein synthesis in HSV-infected cells and inhibits virus replication

Yucca leaf protein (YLP), an inhibitor of tobacco mosaic virus isolated from the leaves of Yucca recurvifolia Salisb., exhibited potent activity against herpes simplex virus type 1 (HSV-1) with no cytotoxicity below 300 μg/ml. The inhibitory dose was varied with the time of addition; 50% effective concentrations (ED 50) of YLP were 3, 19 and 95 μg/ml when YLP exposure was begun 3 h before virus infection, 0 h and 3 h after infection, respectively. This protein also inhibited the multiplication of herpes simplex virus type 2 and human cytomegalovirus. YLP has been shown to have a weak virucidal activity at higher concentrations. Analysis of early events following infection showed that YLP affected viral penetration in HeLa cells but did not interfere with adsorption to the cells. YLP was found to exert strong inhibition of protein synthesis in virus-infected cells but not in uninfected cells. This selective effect can be considered to attribute mainly to the antiviral activity of YLP.

Treatment of premalignancy: prevention of lymphoma in radiation leukemia virus-inoculated mice by cyclosporin A and immunotoxin.

Radiation leukemia virus (RadLV)-induced preleukemic (PL) latency is characterized by the appearance of virus-infected PL cells in the thymus. The survival of these PL cells is dependent upon autostimulation with interleukin 4 (IL-4). We have intervened prophylactically in RadLV-induced preleukemia by using cyclosporin-A (CSA), which inhibits IL-4 production, and an immunotoxin (ITx) that kills PL cells. CSA efficiently inhibited IL-4 secretion from RadLV-induced PL and leukemic cells, and its administration to PL mice caused a significant delay in their death. An ITx consisting of anti-RadLV glycoprotein-70 (gp70) antibody coupled to ricin A chain efficiently inhibited protein synthesis in virus-infected cells in vitro and, when injected into PL mice, also delayed their death. Combined treatment with CSA and ITx prevented 75% of the treated PL mice from developing lymphoma. These results show that the development of malignancy from a premalignant state can be averted by a combination of therapeutic modalities that decrease the size and growth rate of the premalignant cell population.

Modification of membrane permeability during semliki forest virus infection

Modification of membrane permeability has been analyzed in Semliki Forest virus (SFV)-infected cells by means of translation inhibitors not permeable to normal cells. A higher inhibition of protein synthesis in the infected cells is only observed with those antibiotics that do not easily pass the cell membrane, but not with others, permeable to cells, such as anisomycin, cycloheximide, trichodermin, etc. It does not, therefore, seem that the suggestion of M. A. Gray, K. J. Micklem, and C. A. Pasternak [ Eur. J. Biochem. 135, 299–302, (1983)] that protein synthesis in virus-infected cells is more susceptible to translation inhibitors in general, is correct. Both low- and high-molecular weight compounds enter the cell very early during SFV infection. This permeabilization is blocked by compounds known to increase the pH of coated vesicles, such as NH 4Cl and chloroquine. Inhibition of energy production by means of N 3Na and 2′-deoxyglucose also blocks this process. The optimal external pH for this early permeabilization is around 7–8. Acidic pH inhibits the entry of these impermeant antibiotics promoted by SFV. Analysis of 86Rb + content in SFV-infected HeLa cells also indicates that a drastic decline in this cation takes place, in agreement with previous findings, but disagreeing with the previous results. A parallel between the decrease in this cation and the blockade of protein synthesis is apparent, throughout the course of infection. In addition to the early permeabilization that takes place during virus entry, increased entry of hygromycin B and a-sarcin also occurs in SFV-infected cells from 2 to 3 hr postinfection, but not when late viral replication is blocked by means of interferon treatment.

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

HEp-2 cells were infected with herpes simplex virus-1 (HSV-1) or with polio-virus 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 seeds of Gelonium multiflorum, an inhibitor from the seeds of Momordica charantia, dianthin 32, from the leaves of Dianthus caryophyllus (carnation), and PAP-S, from the seeds of Phytolacca 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.

Modification of membrane permeability induced by animal viruses early in infection

The infection of mouse L cells by encephalomyocarditis (EMC) virus renders the cell permeable to several translation inhibitors to which normal cells are impermeable. The modification of membrane permeability to the aminoglycoside antibiotic hygromycin B takes place early in infection during virus adsorption and also at the time when large amounts of viral coat proteins are synthesized. The specific inhibition of protein synthesis in virus-infected cells early in infection is also observed with other translation inhibitors such as anthelmycin, gougerotin, and edeine, indicating that a general permeabilization takes place during virus adsorption. The presence of 1 m M hygromycin B in the culture medium during the first hour of EMC infection strongly reduced the production of new infectious virus. In order to understand the molecular mechanisms involved in membrane permeabilization early during EMC infection, the cells were treated with compounds known to disrupt microtubules and microfilaments and to block pinocytotic processes, e.g., vinblastine, cytochalasin B, and colchicine. However, even in the presence of those compounds the early leakiness phenomenon was still observed. The presence of concanavalin A, which binds to certain cell surface recptors, or inhibitors that block energy production (e.g., FCCP and NaNs) had no significant effects on the modification of membrane permeability. Treatment of human cells with interferon does not prevent the increase in membrane permeability after EMC infection. No viral or cellular protein synthesis is necessary for the early membrane leakiness to occur, suggesting that virions themselves are responsible for this modification. The alteration of permeability to translation inhibitors is also observed after the infection of different mammalian cell lines with different viruses such as vesicular stomatitis virus and Semliki Forest virus, two lipid-enveloped RNA-containing viruses.

Early functions of the genome of herpesvirus: II. Inhibition of the formation of cell-specific polysomes

During the early stages of infection of rabbit kidney cells with pseudorabies virus, when both cell- and virus-specific proteins are being synthesized, two types of polysomes are present in the infected cells: larger virus-specific polysomes with nascent chains of polypeptides characterized by a low lysine: leucine ratio, and smaller cell-specific polysomes with nascent chains of polypeptides characterized by a high lysine: leucine ratio. This finding demonstrates that virus-specific proteins have a lower content of lysine than cell-specific proteins. The difference between cell- and virus-specific proteins in their relative content of amino acids was used to monitor the types of proteins synthesized by the infected cells in studies on the mechanism controlling the shutoff of host protein synthesis in virus-infected cells. Both RNA and protein synthesis are required in the infected cells to effect the inhibition of cell-specific protein synthesis. It was found that a protein (or proteins) is synthesized in virus-infected cells which is responsible for the disappearance of cell-specific polysomes; the message for this protein is transcribed in cells infected in the presence of cycloheximide and is translated after the removal of cycloheximide.